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The use of o-dianisidine for serum haptoglobin electrophoresis using cellulose acetate
Clin. Biochem. 9 (2) 104-105 (1976)
LABORATORY NOTE
The Use of o-Dianisidine for Serum Haptoglobin Electrophoresis Using Cellulose Acetate JOYCE COMPTON, CYNTHIA McCLURE and DEAN P. BONDERMAN Department of Clinical Pathology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA (Accepted January 12, 1976) 0.8-
CLBIA 9, (2) 104-105 (1976) Clin. Bioohem.
0.'/0.6-
Compton, Joyce, McClure, Cynthia, and Bonderman, Dean P. Depart me~tt of Clinical Pathology, India~m U~dversity School of Medicine, Indianapolis, /ndiaua, USA.
THE USE OF O-DIANISIDINE FOR SERUM HAPTOGLOBIN ELECTROPHORESIS USING • CELLULOSE A C E T A T E 1. Due to the carcinogenicity of benzidine, a method by which o-dianisidine is used to stain serum haptoglobin is described. Serum haptoglobin is determined by electrophoresis using cellulose acetate as the medium. 2. A comparison of the two staining systems demonstrates good agreement, o-Dianisidine can be substituted for benzidine without loss of specificity.
TI~E RECENT UNAVAILABILITY OF BENZIDINE due to its c a r c i n o g e n i c i t y has caused the a b a n d o n m e n t of this r e a g e n t in tests t h a t use this redox indicator. One such test is the electrophoresis d e t e r m i n a t i o n of haptoglobins on cellulose acetate. We are c u r r e n t l y u s i n g the system described by Helena `') which employs benzidine. It has been necessary, therefore, to seek an a l t e r n a t i v e to this stain. Owen et al `') has described the use of other s t a i n s such as leuco-malachite green, quanidine, etc., which respond to peroxidase a c t i v i t y u s i n g starch electrophoresis and claimed t h a t o-dianisidine ( 3 - 3 ' - d i m e t h o x y b e n z i d i n e ) was somewhat more sensitive t h a n other s t a i n s i n c l u d i n g benzidine. When we tried to incorporate this s t a i n in our c u r r e n t procedure, we found t h a t the bands formed on the s u r f a c e of the electrophoretic s t r i p d u r i n g color development eluted in the solvent a n d d u r i n g the s u b s e q u e n t methanol dehydration. W y b e n g a et al ~) report a somewhat d i f f e r e n t system of t r e a t m e n t , following electrophoretic separation, t h a n Helena recommends. They did not deproteinize with trichloroacetic acid. This modification allows the s t a i n to incorporate with the intact p r o t e i n m a t e r i a l on the electrophoretic s t r i p
Correspondence: Joyce Compton, M.T.(ASCP), Department of Clinical Pathology, Indiana University Medical Center, 1100 West Michigan Street, Indianapolis, Indiana 46202, U S A
.E
05- _J
J~
--
+ E .c
_
~o4-
~E
~0.3-
~E
o o
-
!
0.2010 - ~ ' ~
J DISTANCE
Fig. 1 - - Methemalbltmin-hemoglobin-haptoglobriu globins-hemoglobin.
hemo-
f o r m i n g an insoluble complex. We, however, f o u n d that the W y b e n g a et al s t a i n system did not work well on our cellulose acetate strips. The s t r i p s do n o t deh y d r a t e properly and s u b s e q u e n t l y cleared unevenly c a u s i n g d i f f i c u l t y in d e n s i t o m e t r y . We find t h a t if we combine the W y b e n g a et al observation on deproteinization with the Helena d e h y d r a t i o n and c l e a r i n g system, o - d i a n i s i d i n e f o r m s a p e r m a n e n t s t a i n on a clear b a c k g r o u n d t h a t can be scanned at 445 n m very adequately ( F i g . l ) . The procedure is as follows: MATERIALS
AND
METHODS
Reagents Absolute methanol. o-Dianisidine stock stain: 150 m g o-dianisidine diluted in 100 ml" mthanol. Acetate buffer: 1.5 mol/liter, p H 4.7. Dissolve 102.2 g sodium acetate tri-hydrate in about 500 ml distilled H20, add 43.5 ml glacial acetic acid, dilute to 1 liter and check pH. Working hydrogen peroxide (3 g/dl): Add 10 ml 30 g/dl hydrogen peroxide to 90 ml distilled H20. Prepare fresh daily. Acetic acid - methanol clearing solution: Add 20 ml glacial acetic acid to 80 ml methanol. Prepare fresh daily. Procedure
Remove cellulose acetate plate(s) from electrophoresis chamber. 1. Immerse plate in methanol for 2 minutes. 2. Prepare working stain: Mix 35 ml, 150 mg/dl o-dianisidine stock; 9 ml distilled H20; 5 ml acetate buffer; add 1 ml working hydrogen peroxide immediately before u s e in a staining dish. Cover with aluminum foil.
o-DIANISIDINE vs BENZIDINE
600
The proposed s t a i n i n g s y s t e m appears to yield r e s u l t s a p p r o x i m a t e l y 20 m g / d l h i g h e r t h a n the b e n z i d i n e system now employed. This may be due to less leaching of the s t a i n by the methanol rinses.
400 -r
We feel t h a t o - d i a n i s i d i n e is an exceptional subs t i t u t e for benzidine with a reduced carcinogenic h a z a r d ' " ~' ~' ~'
~®~X=3.87+1.17(® y) o~ r =0.98
2OO
E
RBFERENCES
/
f
r
I 200
i
I 400
I
I 600
i
I
800
mg/dl Hgb. binding capacity w i t h o- dianisidine
Fig. 2
105
Benzidine-o-dianisidi~e.
3. Place plate in stain for 10 minutes. Cover with aluminum foil. 4. Dehydrate in methanol for 2 minutes. 5. Clear in clearing solution for 2 minutes. 6. Dry in dry heat oven 50-60°C for 8 minutes. 7. Scan using 445 nm filter.
RESULTS A N D DISCUSSION We have compared o - d i a n i s i d i n e and b e n z i d i n e s t a i n i n g systems on f o r t y p a t i e n t s ( F i g . 2). We f i n d t h a t the correlation coefficient is 0.98 i n d i c a t i n g s i m i l a r results a r e o b t a i n e d by both methods. A precision study was done on a frozen pool u s i n g the proposed system. The mean of 20 samples was 102.45 ----4-15.69 mg/d] hemoglobin b i n d i n g capacity. The pool yielded a mean of 81.82 --+ 9.49 mg/d] u s i n g benzidine.
I. Golias, T. L., (1974). Electrophoresis Man~al Helena Laboratories, Beaumont, Texas, Vol. IV (The Haptoglobins). 2. Owcn, J. A., Silberman, H. J., and Got, C., (1958). Detection of hemoglobin, haemoglobin-haptoglobin complexes and other substances with peroxidase activity after zone electrophoresis, Natn.re, 182, 1373. 3. Wybenga, D. R., Ibbott, F. A., and Pileggi, V. J., Determination of haptoglobin in serum. In. Clinical Chemistry: Pri~ziples and Technics, Second Edition, IL J. Henry, D. C. Cannon and J. W. Winkleman, editors; Harper & Row, Hagerstown, N.C., 1974, p 1202. 4. Stokinger, H. E., Chief, Toxicology Branch, Division of Biomedical and Behavioral Science, National Institute for Occupational Health, Cincinnati, Ohio, 45202, Personal communication, December 29, 1975. 5. Gehrmann, G. H., Folger, J. H., and Fleming, A. J., (1949). Occupational tumors of the bladder, Proc. 9th Intl. Congress Ind. Med., 473. 6. Saffiotti, U., Cells, F., Montesano, R., and Sellakatour, A. R., (1966). Induction of bladder cancer in hamsters fed aromatic amines, Ind. Med. and Surg., 564. 7. Sellakumar, A. R., Montesano, R., and Saffiotti, U., (1969). Aromatic amines carcinogenity in hamsters, Proc. Am. Assoc. Cancer Res., 10, 78.
LABORATORY NOTE
The Use of o-Dianisidine for Serum Haptoglobin Electrophoresis Using Cellulose Acetate JOYCE COMPTON, CYNTHIA McCLURE and DEAN P. BONDERMAN Department of Clinical Pathology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA (Accepted January 12, 1976) 0.8-
CLBIA 9, (2) 104-105 (1976) Clin. Bioohem.
0.'/0.6-
Compton, Joyce, McClure, Cynthia, and Bonderman, Dean P. Depart me~tt of Clinical Pathology, India~m U~dversity School of Medicine, Indianapolis, /ndiaua, USA.
THE USE OF O-DIANISIDINE FOR SERUM HAPTOGLOBIN ELECTROPHORESIS USING • CELLULOSE A C E T A T E 1. Due to the carcinogenicity of benzidine, a method by which o-dianisidine is used to stain serum haptoglobin is described. Serum haptoglobin is determined by electrophoresis using cellulose acetate as the medium. 2. A comparison of the two staining systems demonstrates good agreement, o-Dianisidine can be substituted for benzidine without loss of specificity.
TI~E RECENT UNAVAILABILITY OF BENZIDINE due to its c a r c i n o g e n i c i t y has caused the a b a n d o n m e n t of this r e a g e n t in tests t h a t use this redox indicator. One such test is the electrophoresis d e t e r m i n a t i o n of haptoglobins on cellulose acetate. We are c u r r e n t l y u s i n g the system described by Helena `') which employs benzidine. It has been necessary, therefore, to seek an a l t e r n a t i v e to this stain. Owen et al `') has described the use of other s t a i n s such as leuco-malachite green, quanidine, etc., which respond to peroxidase a c t i v i t y u s i n g starch electrophoresis and claimed t h a t o-dianisidine ( 3 - 3 ' - d i m e t h o x y b e n z i d i n e ) was somewhat more sensitive t h a n other s t a i n s i n c l u d i n g benzidine. When we tried to incorporate this s t a i n in our c u r r e n t procedure, we found t h a t the bands formed on the s u r f a c e of the electrophoretic s t r i p d u r i n g color development eluted in the solvent a n d d u r i n g the s u b s e q u e n t methanol dehydration. W y b e n g a et al ~) report a somewhat d i f f e r e n t system of t r e a t m e n t , following electrophoretic separation, t h a n Helena recommends. They did not deproteinize with trichloroacetic acid. This modification allows the s t a i n to incorporate with the intact p r o t e i n m a t e r i a l on the electrophoretic s t r i p
Correspondence: Joyce Compton, M.T.(ASCP), Department of Clinical Pathology, Indiana University Medical Center, 1100 West Michigan Street, Indianapolis, Indiana 46202, U S A
.E
05- _J
J~
--
+ E .c
_
~o4-
~E
~0.3-
~E
o o
-
!
0.2010 - ~ ' ~
J DISTANCE
Fig. 1 - - Methemalbltmin-hemoglobin-haptoglobriu globins-hemoglobin.
hemo-
f o r m i n g an insoluble complex. We, however, f o u n d that the W y b e n g a et al s t a i n system did not work well on our cellulose acetate strips. The s t r i p s do n o t deh y d r a t e properly and s u b s e q u e n t l y cleared unevenly c a u s i n g d i f f i c u l t y in d e n s i t o m e t r y . We find t h a t if we combine the W y b e n g a et al observation on deproteinization with the Helena d e h y d r a t i o n and c l e a r i n g system, o - d i a n i s i d i n e f o r m s a p e r m a n e n t s t a i n on a clear b a c k g r o u n d t h a t can be scanned at 445 n m very adequately ( F i g . l ) . The procedure is as follows: MATERIALS
AND
METHODS
Reagents Absolute methanol. o-Dianisidine stock stain: 150 m g o-dianisidine diluted in 100 ml" mthanol. Acetate buffer: 1.5 mol/liter, p H 4.7. Dissolve 102.2 g sodium acetate tri-hydrate in about 500 ml distilled H20, add 43.5 ml glacial acetic acid, dilute to 1 liter and check pH. Working hydrogen peroxide (3 g/dl): Add 10 ml 30 g/dl hydrogen peroxide to 90 ml distilled H20. Prepare fresh daily. Acetic acid - methanol clearing solution: Add 20 ml glacial acetic acid to 80 ml methanol. Prepare fresh daily. Procedure
Remove cellulose acetate plate(s) from electrophoresis chamber. 1. Immerse plate in methanol for 2 minutes. 2. Prepare working stain: Mix 35 ml, 150 mg/dl o-dianisidine stock; 9 ml distilled H20; 5 ml acetate buffer; add 1 ml working hydrogen peroxide immediately before u s e in a staining dish. Cover with aluminum foil.
o-DIANISIDINE vs BENZIDINE
600
The proposed s t a i n i n g s y s t e m appears to yield r e s u l t s a p p r o x i m a t e l y 20 m g / d l h i g h e r t h a n the b e n z i d i n e system now employed. This may be due to less leaching of the s t a i n by the methanol rinses.
400 -r
We feel t h a t o - d i a n i s i d i n e is an exceptional subs t i t u t e for benzidine with a reduced carcinogenic h a z a r d ' " ~' ~' ~'
~®~X=3.87+1.17(® y) o~ r =0.98
2OO
E
RBFERENCES
/
f
r
I 200
i
I 400
I
I 600
i
I
800
mg/dl Hgb. binding capacity w i t h o- dianisidine
Fig. 2
105
Benzidine-o-dianisidi~e.
3. Place plate in stain for 10 minutes. Cover with aluminum foil. 4. Dehydrate in methanol for 2 minutes. 5. Clear in clearing solution for 2 minutes. 6. Dry in dry heat oven 50-60°C for 8 minutes. 7. Scan using 445 nm filter.
RESULTS A N D DISCUSSION We have compared o - d i a n i s i d i n e and b e n z i d i n e s t a i n i n g systems on f o r t y p a t i e n t s ( F i g . 2). We f i n d t h a t the correlation coefficient is 0.98 i n d i c a t i n g s i m i l a r results a r e o b t a i n e d by both methods. A precision study was done on a frozen pool u s i n g the proposed system. The mean of 20 samples was 102.45 ----4-15.69 mg/d] hemoglobin b i n d i n g capacity. The pool yielded a mean of 81.82 --+ 9.49 mg/d] u s i n g benzidine.
I. Golias, T. L., (1974). Electrophoresis Man~al Helena Laboratories, Beaumont, Texas, Vol. IV (The Haptoglobins). 2. Owcn, J. A., Silberman, H. J., and Got, C., (1958). Detection of hemoglobin, haemoglobin-haptoglobin complexes and other substances with peroxidase activity after zone electrophoresis, Natn.re, 182, 1373. 3. Wybenga, D. R., Ibbott, F. A., and Pileggi, V. J., Determination of haptoglobin in serum. In. Clinical Chemistry: Pri~ziples and Technics, Second Edition, IL J. Henry, D. C. Cannon and J. W. Winkleman, editors; Harper & Row, Hagerstown, N.C., 1974, p 1202. 4. Stokinger, H. E., Chief, Toxicology Branch, Division of Biomedical and Behavioral Science, National Institute for Occupational Health, Cincinnati, Ohio, 45202, Personal communication, December 29, 1975. 5. Gehrmann, G. H., Folger, J. H., and Fleming, A. J., (1949). Occupational tumors of the bladder, Proc. 9th Intl. Congress Ind. Med., 473. 6. Saffiotti, U., Cells, F., Montesano, R., and Sellakatour, A. R., (1966). Induction of bladder cancer in hamsters fed aromatic amines, Ind. Med. and Surg., 564. 7. Sellakumar, A. R., Montesano, R., and Saffiotti, U., (1969). Aromatic amines carcinogenity in hamsters, Proc. Am. Assoc. Cancer Res., 10, 78.