Free Radical Biology & Medicine, Vol. 10, pp. 507-510, 1990 Printed in the USA. All rights reserved.
0891-5849/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
- Original • "Contribution COMPARISON OF FOUR INDIRECT METHODS FOR FLUID SUPEROXIDE DISMUTASE ACTIVITIES
ROBERT A. DISILVESTRO, CHRISTINA DAVID, and ELIZABETHA. DAVID Department of Foods & Nutrition, Purdue University, West Lafayette, IN 47907, USA (Received 7 June 1990; Revised and Accepted 7 September 1990)
Abstract--Relatively small sample dilutions could render fluid extracellular (EC) superoxide dismutase (SOD) activity assays more subject to interfering compounds than tissue SOD assays. Highly variable relative SOD activities were obtained when comparing four indirect assays for several fluid samples (human plasma, human synovial fluid, and plasma from healthy or inflamed rats). Analysis of rat plasma fractionated with Sephadex G-150 showed that each assay (three xanthine oxidase based assays plus a modified pyrogallol assay) detected apparent SOD activity almost entirely at the same molecular weight as rat lung EC SOD. However, unfractionated fluid samples caused interferences with the xanthine oxidase based SOD assays, though not with the pyrogallol method. Examples of interference were stimulation of xanthine oxidase activity, color formation without xanthine oxidase, color formation despite excess Cu-Zn SOD addition, and absorbance changes with cyanide inhibition of EC SOD that were above or below blank values. In summary, relative fluid SOD values depended on the assay used, and a modified pyrogallol assay was not subject to several interferences found for three xanthine oxidase based assays of fluid SOD activity. Keywords--Superoxide dismutase, Plasma, Synovial fluid
subjects. However, use of different SOD assays could also produce the diverse results. Such behavior has been shown in rats by DiSilvestro. 6"7 Experimental inflammation raises serum SOD activity levels by one assay, 6 but decreases them by another. 7 The present study compared four indirect SOD assays for relative values derived for various fluid samples, and tested for several types of assay interference.
INTRODUCTION
Recently, an extracellular protein with superoxide dismutase (SOD) activity has been found in tissues and biological fluids. 1,2 Activity concentrations of extracellular (EC) SOD in fluid or tissue samples are much lower than cellular levels of either of the two intracellular SOD enzymes. 2 This can create problems for fluid SOD activity assays since low sample dilutions cause high assay concentrations of potential interfering agents. Most studies of SOD enzymes utilize so called indirect assays. SOD competes with an indicator molecule for superoxide radical. 3 These assays can be subject to interferences, even for tissue measures which involve less sample dilution than fluid assays. 3 Already, studies of fluid SOD have produced conflicting results. Synovial fluid SOD activities in rheumatoid arthritics are reported as higher than synovial fluid from osteoarthritics. 4 Elsewhere, synovial fluid SOD activities in rheumatoid arthritics are reported as below those from people with nonarthritic injuries. 5 This conflict might arise partly from variations in the study
MATERIALS AND METHODS
Protein sources
Bovine Cu-Zn SOD was obtained from DDI Laboratories, Mountain View, CA. Bovine xanthine oxidase (Type II grade) and human ceruloplasmin were purchased from Sigma Chemical, St. Louis, MO, and apotransferrin bought from ICN Biochemicals, Cleveland, OH.
Fluid samples
Rat plasma used to study assay interferences was pooled from 10 male, Wistar rats weighing between 250 and 400 g. Inflammation studies used six rats (250 to 300 g), half injected with 0.1 mL turpentine (im, leg) 3
Address correspondence to Robert A. DiSilvestro, Department of Human Nutrition & Food Management, Ohio State University, 1787 Neil Ave, 265 Campbell Hall, Columbus, OH 43210. 507
R.A. DISILVESTROet al.
508
Table 1. Features of SOD Assays. Assay & Reference NBT8 Nitrite9 Cyt. Ct° Pyrogallo112
Superoxide Generator Detector Xanthine Ox. Xanthine Xanthine Ox. Hypoxanthine XanthineOx. Xanthine Pyrogallol
NBT
Parameter
Blank Value
Sample Volume0xL)
A55on m
0.2
Endpoint A55onm/30s Kinetic
0.012
500a 250b 100a 50b Vary~
A32onm/30s
0.016 b
Varyc
Kinetic
0.008a
A56on m
0.2
Endpoint Sulfanilic Acid CytochromeC Pyrogallol
Abbreviations:nitrobluetetrazolium--NBT;oxidase--ox.; cytochrome C--Cyt. C. "For human plasma. bFor synovialfluid and rat plasma. CVolumewas varied until 50% inhibitionwas obtained.
days before sacrifice. Human plasma was reconstituted from a lyophilized pool sold by Calbiochem, San Diego, CA. Synovial fluid was from six rheumatoid arthritis patients and was treated with hyaluronidase to reduce viscosity. 5
E n z y m e assays
SOD enzyme assays are summarized in Table 1. The original pyrogallol method ~ was modified 12 to increase sensitivity. For both end point methods, 100% inhibition was the absorbance reading obtained for Cu-Zn SOD standard at levels giving maximal inhibition. Maximal inhibition was the percent inhibition which was not increased by further raising Cu-Zn SOD concentrations. Ceruloplasmin activity was measured as p-phenylenediamine oxidase. 7 A unit was absorbance at 520nm after 15 rain minus absorbance from sample added to the assay mix after stopping the reaction with azide. Xanthine oxidase activity was assessed by conversion of xanthine to uric acid using absorbance at 295nm.13
RESULTS
Four SOD assays gave very different relative activity values when comparing rat plasma to human plasma
to human synovial fluid (Table 2). The four SOD methods also gave very different relative activity values when comparing normal rat plasma to plasma from rats undergoing experimental inflammation (Table 3). The rise in ceruloplasmin activities with turpentine confirmed the occurrence of an inflammation-induced acute phase response. 14 Tables 2 and 3 clearly demonstrate that the SOD assays tested could not each be providing accurate relative fluid SOD values. The discrepancy did not result from detection of apparent SOD activity at a molecular weight not corresponding to known SOD enzymes. Previously, this laboratory, 7 using gel filtration with Sephadex G-150, found pyrogallol detectable rat serum SOD activity almost exclusively at the same molecular weight as rat lung EC SOD. A very small activity peak is seen in the Cu-Zn SOD region. In the present study, the same pattern was found for fractionated rat plasma using any of the assays studied here (data not shown). The balance of the study examined whether various interferences potentially affect the SOD assays. First, biological fluid was found to stimulate activity of xanthine oxidase, the superoxide generator for three of the assays (Table 4). Xanthine was the substrate, but the problem persisted when rat plasma was tested with byTable 3. Effects of Inflammationon Rat Plasma SOD Levels Control
Table 2. Sample Values for Various SOD Methods
Assay NBT Endpoint Nitrite Endpoint Cytochrome C Pyrogallol
Rat Human Plasma Plasma SynovialFI (Units/dL) (Units/dL) (Units/dL) Ratios 4,130 10,646 2,632 7,692
32 2,254 333 800
253 1,061 1,000 1,667
129:1:8 10:2:1 8:1:3 10:1:2
Results were duplicate means of pooled samples. Units were the sample amountgiving 50% inhibitionof the uninhibitedreaction.
Ceruloplasmin Units/dL SOD Units/dL NBT Nitrite Cytochrome C Pyrogallol
84 + 4,047 11,701 2,534 8,772
+__ 975 ___ 1,557 _+_ 551 ___ 895
+ Inflammation
208 -+ 8 6,200 24,512 7,392 5,000
-+ 782 _+ 543 _+ 1,285 -+ 250
Inflammationwas inducedby 0.1 mL turpentine(im, leg) 3 days before sacrifice. Values are means - SD for three rats. All inflammation values were significantlydifferent from controls (p < 0.01, Student's t test).
Fluid superoxide dismutase assays Table 4. Effects of Fluids on Xanthine Oxidase Activity Change in Absorbance295n m Per Min × 103 Fluid None Rat Plasma Human Plasma Synovial Fluid
- XO
0 3 1 2
-+ 0 -+ 1 -+ 1 --- 1
+XO
67 112 I01 98
_+ 1 __- 6 ___ 5 --+ 5
Results are the means of three trials _+ SD. All assays were carfled out using 70 I~L of fluid. All values in the + XO column were significantly different from values in the - XO column (p 10% above a no interference value (absorbance of assay reagents plus absorbance of samples diluted to assay volume with water). High or low designations required > 10% deviation from cyanide blank value + absorbance from sample diluted to assay volume with water. Sample volumes were those used for Table I. aNot applicable.
here, a modified pyrogallol assay was more accurate than three xanthine oxidase based methods. The latter methods were subject to interferences which could increase or decreease apparent SOD values. Therefore, SOD values would represent actual SOD activity plus the net effect of positive and negative interferences. Conceivably, sample donor variables (i.e., inflammatory state) could cause variable degrees of interference. Also, the extent of any interference depends on sample volume, which can vary between assay and sample (Table 1). This study supported the assertion that use of different SOD assays contributed to the conflicting results for the above mentioned studies on rheumatoid arthritis patients. 4"5 One study used the cytochrome C method while the other used a direct SOD method. The present study did not compare synovial fluid from rheumatoid arthritis to samples from people without inflammatory diseases. However, for the pyrogallol assay, inflammation reduced rat plasma SOD activities, an effect similar to that of rheumatoid arthritis on synovial fluid SOD measured by direct SOD assay. 5 This study, like most previous ones on fluid SOD, used samples without pretreatment such as dialysis or protein precipitation. In one study from this laboratory, rat serum was dialyzed before pyrogallol SOD assay. 7 Subsequently, readings were found unaffected by dialysis (DiSilvestro, R.A., unpublished results). Another
510
R.A. DISILVESTROet al.
g r o u p h a s applied the N B T e n d p o i n t a s s a y to s e r u m treated w i t h e t h a n o l : c h l o r o f o r m to p r e c i p i t a t e s o m e n o n S O D proteins. 9 P o s s i b l y , s o m e s a m p l e t r e a t m e n t ( s ) c o u l d b e u s e d to r e m o v e c e r t a i n i n t e r f e r e n c e s f r o m the xant h i n e o x i d a s e b a s e d S O D assays. In c o n c l u s i o n , this study suggests t h r e e p o t e n t i a l a p p r o a c h e s for i n d i r e c t S O D assay o f fluids. T h e m o d i f i e d p y r o g a l l o l a s s a y c a n b e used, s a m p l e t r e a t m e n t s c a n b e d e r i v e d to e l i m i n a t e fluid i n t e r f e r e n c e s w i t h x a n t h i n e o x i d a s e b a s e d m e t h ods, or m a t h e m a t i c a l c o r r e c t i o n f o r m u l a s c a n b e develo p e d for t h e s e i n t e r f e r e n c e s . Acknowledgements -- The authors wish to recognize the fine techni-
cal assistance of Joy Frederici, Feili Yang, and Lisa Schaller. This work was supported in part by a grant from the Indiana Chapter of the Arthritis Foundation and by the Purdue Agricultural Experiment Station High School Summer Apprentice Program.
REFERENCES
1. Hassan, H.M. Biosynthesis and regulation of superoxide dismutases. Free Radic. Biol. Med. 5:377-385; 1988. 2. Marklund, S.L. Human copper-containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. USA 79:76347638; 1982. 3. Marklund, S.L.; Holme, E.; Hellner, L. Superoxide dismutase in extracellular fluids. Clin. Chim. Acta. 126:41-51; 1982. 4. Bannister, J.V.; Calabrese, L. Assays for superoxide dismutase.
Meth. Biochem. Anal. 32:279-313; 1987. 5. Igari, T.; Kaneda, H.; Horiuchi, S.; Ono, S. A remarkable increase of superoxide dismutase activity in synovial fluid of patients with rheumatoid arthritis. Clin. Orthoped. Rel. Res. 162: 282-287; 1982. 6. Marklund, S.L.; Bjelle, A.; Elmqvist, L.G. Superoxide dismutase isoenzymes of the synovial fluid in rheumatoid arthritis and in reactive arthritides. Ann, Rheum. Dis. 45:847-851; 1986. 7. DiSilvestro, R.A. Influence of diet and inflammation on serum copper enzymes. In: Sorenson, J.R.J., ed. Biology of copper complexes. Clifton, NJ: Humana Press; 1987:59-66. 8. DiSilvestro, R.A. Influence of copper intake and inflammation on rat serum superoxide dismutase activity levels. J. Nutr. 118: 474-479; 1988. 9. Sun, Y.; Oberley, L.W.; Li, Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34:497-500; 1988. 10. Oyanagui, Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal. Biochem. 142: 290-296; 1984. 11. Fridovich, I. Cytochrome c. In: Greenwald, R.A., ed. Handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985:213-215. 12. Marklund, S.; Marklund, G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469-474; 1974. 13. Prohaska, J. Changes in tissue growth, concentrations of copper, iron, cytochrome oxidase and superoxide dismutase subsequent to dietary or genetic copper deficiency in mice. J. Nutr. 113:21482158; 1983. 14. Rajagopalan, K.V. Purification of bovine milk xanthine oxidase. In: Greenwald, R.A., ed. Handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985;21-23. 15. Cousins, R.J. Absorption, transport, and heptatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65:238-309; 1985,
0891-5849/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
- Original • "Contribution COMPARISON OF FOUR INDIRECT METHODS FOR FLUID SUPEROXIDE DISMUTASE ACTIVITIES
ROBERT A. DISILVESTRO, CHRISTINA DAVID, and ELIZABETHA. DAVID Department of Foods & Nutrition, Purdue University, West Lafayette, IN 47907, USA (Received 7 June 1990; Revised and Accepted 7 September 1990)
Abstract--Relatively small sample dilutions could render fluid extracellular (EC) superoxide dismutase (SOD) activity assays more subject to interfering compounds than tissue SOD assays. Highly variable relative SOD activities were obtained when comparing four indirect assays for several fluid samples (human plasma, human synovial fluid, and plasma from healthy or inflamed rats). Analysis of rat plasma fractionated with Sephadex G-150 showed that each assay (three xanthine oxidase based assays plus a modified pyrogallol assay) detected apparent SOD activity almost entirely at the same molecular weight as rat lung EC SOD. However, unfractionated fluid samples caused interferences with the xanthine oxidase based SOD assays, though not with the pyrogallol method. Examples of interference were stimulation of xanthine oxidase activity, color formation without xanthine oxidase, color formation despite excess Cu-Zn SOD addition, and absorbance changes with cyanide inhibition of EC SOD that were above or below blank values. In summary, relative fluid SOD values depended on the assay used, and a modified pyrogallol assay was not subject to several interferences found for three xanthine oxidase based assays of fluid SOD activity. Keywords--Superoxide dismutase, Plasma, Synovial fluid
subjects. However, use of different SOD assays could also produce the diverse results. Such behavior has been shown in rats by DiSilvestro. 6"7 Experimental inflammation raises serum SOD activity levels by one assay, 6 but decreases them by another. 7 The present study compared four indirect SOD assays for relative values derived for various fluid samples, and tested for several types of assay interference.
INTRODUCTION
Recently, an extracellular protein with superoxide dismutase (SOD) activity has been found in tissues and biological fluids. 1,2 Activity concentrations of extracellular (EC) SOD in fluid or tissue samples are much lower than cellular levels of either of the two intracellular SOD enzymes. 2 This can create problems for fluid SOD activity assays since low sample dilutions cause high assay concentrations of potential interfering agents. Most studies of SOD enzymes utilize so called indirect assays. SOD competes with an indicator molecule for superoxide radical. 3 These assays can be subject to interferences, even for tissue measures which involve less sample dilution than fluid assays. 3 Already, studies of fluid SOD have produced conflicting results. Synovial fluid SOD activities in rheumatoid arthritics are reported as higher than synovial fluid from osteoarthritics. 4 Elsewhere, synovial fluid SOD activities in rheumatoid arthritics are reported as below those from people with nonarthritic injuries. 5 This conflict might arise partly from variations in the study
MATERIALS AND METHODS
Protein sources
Bovine Cu-Zn SOD was obtained from DDI Laboratories, Mountain View, CA. Bovine xanthine oxidase (Type II grade) and human ceruloplasmin were purchased from Sigma Chemical, St. Louis, MO, and apotransferrin bought from ICN Biochemicals, Cleveland, OH.
Fluid samples
Rat plasma used to study assay interferences was pooled from 10 male, Wistar rats weighing between 250 and 400 g. Inflammation studies used six rats (250 to 300 g), half injected with 0.1 mL turpentine (im, leg) 3
Address correspondence to Robert A. DiSilvestro, Department of Human Nutrition & Food Management, Ohio State University, 1787 Neil Ave, 265 Campbell Hall, Columbus, OH 43210. 507
R.A. DISILVESTROet al.
508
Table 1. Features of SOD Assays. Assay & Reference NBT8 Nitrite9 Cyt. Ct° Pyrogallo112
Superoxide Generator Detector Xanthine Ox. Xanthine Xanthine Ox. Hypoxanthine XanthineOx. Xanthine Pyrogallol
NBT
Parameter
Blank Value
Sample Volume0xL)
A55on m
0.2
Endpoint A55onm/30s Kinetic
0.012
500a 250b 100a 50b Vary~
A32onm/30s
0.016 b
Varyc
Kinetic
0.008a
A56on m
0.2
Endpoint Sulfanilic Acid CytochromeC Pyrogallol
Abbreviations:nitrobluetetrazolium--NBT;oxidase--ox.; cytochrome C--Cyt. C. "For human plasma. bFor synovialfluid and rat plasma. CVolumewas varied until 50% inhibitionwas obtained.
days before sacrifice. Human plasma was reconstituted from a lyophilized pool sold by Calbiochem, San Diego, CA. Synovial fluid was from six rheumatoid arthritis patients and was treated with hyaluronidase to reduce viscosity. 5
E n z y m e assays
SOD enzyme assays are summarized in Table 1. The original pyrogallol method ~ was modified 12 to increase sensitivity. For both end point methods, 100% inhibition was the absorbance reading obtained for Cu-Zn SOD standard at levels giving maximal inhibition. Maximal inhibition was the percent inhibition which was not increased by further raising Cu-Zn SOD concentrations. Ceruloplasmin activity was measured as p-phenylenediamine oxidase. 7 A unit was absorbance at 520nm after 15 rain minus absorbance from sample added to the assay mix after stopping the reaction with azide. Xanthine oxidase activity was assessed by conversion of xanthine to uric acid using absorbance at 295nm.13
RESULTS
Four SOD assays gave very different relative activity values when comparing rat plasma to human plasma
to human synovial fluid (Table 2). The four SOD methods also gave very different relative activity values when comparing normal rat plasma to plasma from rats undergoing experimental inflammation (Table 3). The rise in ceruloplasmin activities with turpentine confirmed the occurrence of an inflammation-induced acute phase response. 14 Tables 2 and 3 clearly demonstrate that the SOD assays tested could not each be providing accurate relative fluid SOD values. The discrepancy did not result from detection of apparent SOD activity at a molecular weight not corresponding to known SOD enzymes. Previously, this laboratory, 7 using gel filtration with Sephadex G-150, found pyrogallol detectable rat serum SOD activity almost exclusively at the same molecular weight as rat lung EC SOD. A very small activity peak is seen in the Cu-Zn SOD region. In the present study, the same pattern was found for fractionated rat plasma using any of the assays studied here (data not shown). The balance of the study examined whether various interferences potentially affect the SOD assays. First, biological fluid was found to stimulate activity of xanthine oxidase, the superoxide generator for three of the assays (Table 4). Xanthine was the substrate, but the problem persisted when rat plasma was tested with byTable 3. Effects of Inflammationon Rat Plasma SOD Levels Control
Table 2. Sample Values for Various SOD Methods
Assay NBT Endpoint Nitrite Endpoint Cytochrome C Pyrogallol
Rat Human Plasma Plasma SynovialFI (Units/dL) (Units/dL) (Units/dL) Ratios 4,130 10,646 2,632 7,692
32 2,254 333 800
253 1,061 1,000 1,667
129:1:8 10:2:1 8:1:3 10:1:2
Results were duplicate means of pooled samples. Units were the sample amountgiving 50% inhibitionof the uninhibitedreaction.
Ceruloplasmin Units/dL SOD Units/dL NBT Nitrite Cytochrome C Pyrogallol
84 + 4,047 11,701 2,534 8,772
+__ 975 ___ 1,557 _+_ 551 ___ 895
+ Inflammation
208 -+ 8 6,200 24,512 7,392 5,000
-+ 782 _+ 543 _+ 1,285 -+ 250
Inflammationwas inducedby 0.1 mL turpentine(im, leg) 3 days before sacrifice. Values are means - SD for three rats. All inflammation values were significantlydifferent from controls (p < 0.01, Student's t test).
Fluid superoxide dismutase assays Table 4. Effects of Fluids on Xanthine Oxidase Activity Change in Absorbance295n m Per Min × 103 Fluid None Rat Plasma Human Plasma Synovial Fluid
- XO
0 3 1 2
-+ 0 -+ 1 -+ 1 --- 1
+XO
67 112 I01 98
_+ 1 __- 6 ___ 5 --+ 5
Results are the means of three trials _+ SD. All assays were carfled out using 70 I~L of fluid. All values in the + XO column were significantly different from values in the - XO column (p 10% above a no interference value (absorbance of assay reagents plus absorbance of samples diluted to assay volume with water). High or low designations required > 10% deviation from cyanide blank value + absorbance from sample diluted to assay volume with water. Sample volumes were those used for Table I. aNot applicable.
here, a modified pyrogallol assay was more accurate than three xanthine oxidase based methods. The latter methods were subject to interferences which could increase or decreease apparent SOD values. Therefore, SOD values would represent actual SOD activity plus the net effect of positive and negative interferences. Conceivably, sample donor variables (i.e., inflammatory state) could cause variable degrees of interference. Also, the extent of any interference depends on sample volume, which can vary between assay and sample (Table 1). This study supported the assertion that use of different SOD assays contributed to the conflicting results for the above mentioned studies on rheumatoid arthritis patients. 4"5 One study used the cytochrome C method while the other used a direct SOD method. The present study did not compare synovial fluid from rheumatoid arthritis to samples from people without inflammatory diseases. However, for the pyrogallol assay, inflammation reduced rat plasma SOD activities, an effect similar to that of rheumatoid arthritis on synovial fluid SOD measured by direct SOD assay. 5 This study, like most previous ones on fluid SOD, used samples without pretreatment such as dialysis or protein precipitation. In one study from this laboratory, rat serum was dialyzed before pyrogallol SOD assay. 7 Subsequently, readings were found unaffected by dialysis (DiSilvestro, R.A., unpublished results). Another
510
R.A. DISILVESTROet al.
g r o u p h a s applied the N B T e n d p o i n t a s s a y to s e r u m treated w i t h e t h a n o l : c h l o r o f o r m to p r e c i p i t a t e s o m e n o n S O D proteins. 9 P o s s i b l y , s o m e s a m p l e t r e a t m e n t ( s ) c o u l d b e u s e d to r e m o v e c e r t a i n i n t e r f e r e n c e s f r o m the xant h i n e o x i d a s e b a s e d S O D assays. In c o n c l u s i o n , this study suggests t h r e e p o t e n t i a l a p p r o a c h e s for i n d i r e c t S O D assay o f fluids. T h e m o d i f i e d p y r o g a l l o l a s s a y c a n b e used, s a m p l e t r e a t m e n t s c a n b e d e r i v e d to e l i m i n a t e fluid i n t e r f e r e n c e s w i t h x a n t h i n e o x i d a s e b a s e d m e t h ods, or m a t h e m a t i c a l c o r r e c t i o n f o r m u l a s c a n b e develo p e d for t h e s e i n t e r f e r e n c e s . Acknowledgements -- The authors wish to recognize the fine techni-
cal assistance of Joy Frederici, Feili Yang, and Lisa Schaller. This work was supported in part by a grant from the Indiana Chapter of the Arthritis Foundation and by the Purdue Agricultural Experiment Station High School Summer Apprentice Program.
REFERENCES
1. Hassan, H.M. Biosynthesis and regulation of superoxide dismutases. Free Radic. Biol. Med. 5:377-385; 1988. 2. Marklund, S.L. Human copper-containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. USA 79:76347638; 1982. 3. Marklund, S.L.; Holme, E.; Hellner, L. Superoxide dismutase in extracellular fluids. Clin. Chim. Acta. 126:41-51; 1982. 4. Bannister, J.V.; Calabrese, L. Assays for superoxide dismutase.
Meth. Biochem. Anal. 32:279-313; 1987. 5. Igari, T.; Kaneda, H.; Horiuchi, S.; Ono, S. A remarkable increase of superoxide dismutase activity in synovial fluid of patients with rheumatoid arthritis. Clin. Orthoped. Rel. Res. 162: 282-287; 1982. 6. Marklund, S.L.; Bjelle, A.; Elmqvist, L.G. Superoxide dismutase isoenzymes of the synovial fluid in rheumatoid arthritis and in reactive arthritides. Ann, Rheum. Dis. 45:847-851; 1986. 7. DiSilvestro, R.A. Influence of diet and inflammation on serum copper enzymes. In: Sorenson, J.R.J., ed. Biology of copper complexes. Clifton, NJ: Humana Press; 1987:59-66. 8. DiSilvestro, R.A. Influence of copper intake and inflammation on rat serum superoxide dismutase activity levels. J. Nutr. 118: 474-479; 1988. 9. Sun, Y.; Oberley, L.W.; Li, Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34:497-500; 1988. 10. Oyanagui, Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal. Biochem. 142: 290-296; 1984. 11. Fridovich, I. Cytochrome c. In: Greenwald, R.A., ed. Handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985:213-215. 12. Marklund, S.; Marklund, G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469-474; 1974. 13. Prohaska, J. Changes in tissue growth, concentrations of copper, iron, cytochrome oxidase and superoxide dismutase subsequent to dietary or genetic copper deficiency in mice. J. Nutr. 113:21482158; 1983. 14. Rajagopalan, K.V. Purification of bovine milk xanthine oxidase. In: Greenwald, R.A., ed. Handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985;21-23. 15. Cousins, R.J. Absorption, transport, and heptatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65:238-309; 1985,
