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Rapid electrophoresis of alkaline phosphatase isoenzymes
BRIEF
303
NOTES
Rapid electrophoresis of alkaline phosphatase isoenzymes Several methods for studying the different isoenzyme fractions of alkaline phosphatase are in use. Heat stability1,urea2- or L-phenylalanines-inhibition methods destroy one fraction in order to measure another, and therefore cannot analyse mixtures of two or more fractions. Electrophoresis using cellulose acetate4, starch gels, agara3’, or acrylamide gels+, separates complex mixtures, but the methods so far described are either time-consuming or unreliable. The method we describe is simple and rapid, and can be applied to the separation and identification of isoenzymes of liver, bone, placental and intestinal origin in any routine laboratory. METHODS
I. Electrophoresis
The electrophoresis tank was constructed according to Wiemee, with petroleum ether as the coolant. The buffer used for electrophoresis was 0.05 M barbitone at pH 8.4 with 0.1% thiomersalate. Standard microscopy glass slides (0.9 mm thick) were coated with 1% Difco “Special Agar Noble”* in buffer to a thickness of 1.0 mm. Slits, 4 mm wide, were made in the agar by inserting strips of Whatman No. 54 filter paper, and 2-4 ~1 of serum inserted according to the activity of the total serum alkaline phosphatase. Electrophoresis was performed for 25 min with a current of 30 mA per slide and a gradient of 15 V/cm. 2. Staining
In the enzyme-substrate reaction, p-naphthol is released and is coupled with the diazonium salt Fast Blue RR to give red zones of phosphatase activity. Solution I: 0.1 M sodium carbonate/bicarbonate Solution II : 0.015 M magnesium sulphate.
buffer, pH 10.0 (Kinglo).
Immediately before use, 3 mg of Fast Blue RR * * is dissolved in 6 ml of solution I, and 3 mg of sodium fi-naphthyl phosphate * * dissolved in 6 ml of solution II. The two solutions are then mixed, poured onto the unfixed’slide, and left at room temperature for 15 min. When colour development is complete, the patterns are fixed and washed in successive changes of 5% acetic acid, and dried overnight between slieets of Whatman No. I filter paper (Fig. I). The liver isoenzyme moves fastest, and is seen in the cr,-macroglobulin region. The bone isoenzyme is slower and more diffuse, and shows as a broad band between the cr,-globulin and the origin. The placental isoenzyme moves as one or two fractions close to the origin, and is present in sera from pregnant women. The intestinal isoenzyme is the slowest of all, and migrates in the B-r region. Mixtures of isoenzymes can be clearly separated, and zones of activity are visible from sera containing as little as IO King-Armstrong units per IOO ml. The method differs from that originally described by Wiemeg. The sodium carbonate-bicarbonate buffer appeared to be superior to his borate buffer. Our * Difco Laboratories Inc., Detroit, Mich., U.S.A. * * K and K Laboratories Inc.. Plainview, N.Y., U.S.A. Cltn. Chim.
Acta,
32 (1971)
303-304
BRIEF NOTES
304
Fig. I. Alkaline phosphatase isoenzymes in human serum, separated by agar gel electrophoresis isoenzymes of I. liver 2. bone 3. placental and 4. intestinal origin.
:
aqueous staining technique was simpler than preparation of the buffer, substrate and colour reagent in agar gel. Colour development proceeded more rapidly at room temperature than at ~7~. This method was compared with the agar gel method of Yang’, in which a much higher voltage is made possible by active recycling of coolant. In our hands both techniques gave similar patterns. Attempts to stain the isoenzyme patterns by the “lead conversion” methods gave very poor results. Depnrtntent of Child Health, Kirg’s College Hospital, Lo&on, S. E.5 (U.K.)
J. R. RAWSTRON S. H. NG
I S. POSEN,Ii. C. NEALE AND,J. S. CLUBB. Ann. Internal Med., 62 (1965) 1234. 2 M. HORNE, C. J. CORNISH AND S. POSEN, J. Lab. Clin. Med., 72 (1968) 905. 3 W. H. FISHMAN AND N. K. GHOSH, Advan. Clin. Chem., IO (1967) 255. 4 N. H. KORNER, J. C&n. Pathol, 15 (1962) 195. 5 bl. A. NEWTON, @art J. Med., 36 (1967) 17. 0 R. J. WIEME, Agar Grl Eelectrophoresis, Elsevier, Amsterdam, 1965, pp. 68-76, 163-164 7 J. M. YONG, J. Clin. Pathol, zo (1967) 647. 8 J. M. ALLEN AND G. J. HYNICK, J. Histochem. Cytochem., II (1963) 169. g M. M. KAPLAN AND L. ROGERS, Lancet, ii (1969) Iozg. IO E. J. KING, Micro-analysis in Medical Biochemistry, Churchill, London, 1951. p. 72.
Received Clin. Chint.
October Acta,
19, 1970
32 (197’)
303-304
303
NOTES
Rapid electrophoresis of alkaline phosphatase isoenzymes Several methods for studying the different isoenzyme fractions of alkaline phosphatase are in use. Heat stability1,urea2- or L-phenylalanines-inhibition methods destroy one fraction in order to measure another, and therefore cannot analyse mixtures of two or more fractions. Electrophoresis using cellulose acetate4, starch gels, agara3’, or acrylamide gels+, separates complex mixtures, but the methods so far described are either time-consuming or unreliable. The method we describe is simple and rapid, and can be applied to the separation and identification of isoenzymes of liver, bone, placental and intestinal origin in any routine laboratory. METHODS
I. Electrophoresis
The electrophoresis tank was constructed according to Wiemee, with petroleum ether as the coolant. The buffer used for electrophoresis was 0.05 M barbitone at pH 8.4 with 0.1% thiomersalate. Standard microscopy glass slides (0.9 mm thick) were coated with 1% Difco “Special Agar Noble”* in buffer to a thickness of 1.0 mm. Slits, 4 mm wide, were made in the agar by inserting strips of Whatman No. 54 filter paper, and 2-4 ~1 of serum inserted according to the activity of the total serum alkaline phosphatase. Electrophoresis was performed for 25 min with a current of 30 mA per slide and a gradient of 15 V/cm. 2. Staining
In the enzyme-substrate reaction, p-naphthol is released and is coupled with the diazonium salt Fast Blue RR to give red zones of phosphatase activity. Solution I: 0.1 M sodium carbonate/bicarbonate Solution II : 0.015 M magnesium sulphate.
buffer, pH 10.0 (Kinglo).
Immediately before use, 3 mg of Fast Blue RR * * is dissolved in 6 ml of solution I, and 3 mg of sodium fi-naphthyl phosphate * * dissolved in 6 ml of solution II. The two solutions are then mixed, poured onto the unfixed’slide, and left at room temperature for 15 min. When colour development is complete, the patterns are fixed and washed in successive changes of 5% acetic acid, and dried overnight between slieets of Whatman No. I filter paper (Fig. I). The liver isoenzyme moves fastest, and is seen in the cr,-macroglobulin region. The bone isoenzyme is slower and more diffuse, and shows as a broad band between the cr,-globulin and the origin. The placental isoenzyme moves as one or two fractions close to the origin, and is present in sera from pregnant women. The intestinal isoenzyme is the slowest of all, and migrates in the B-r region. Mixtures of isoenzymes can be clearly separated, and zones of activity are visible from sera containing as little as IO King-Armstrong units per IOO ml. The method differs from that originally described by Wiemeg. The sodium carbonate-bicarbonate buffer appeared to be superior to his borate buffer. Our * Difco Laboratories Inc., Detroit, Mich., U.S.A. * * K and K Laboratories Inc.. Plainview, N.Y., U.S.A. Cltn. Chim.
Acta,
32 (1971)
303-304
BRIEF NOTES
304
Fig. I. Alkaline phosphatase isoenzymes in human serum, separated by agar gel electrophoresis isoenzymes of I. liver 2. bone 3. placental and 4. intestinal origin.
:
aqueous staining technique was simpler than preparation of the buffer, substrate and colour reagent in agar gel. Colour development proceeded more rapidly at room temperature than at ~7~. This method was compared with the agar gel method of Yang’, in which a much higher voltage is made possible by active recycling of coolant. In our hands both techniques gave similar patterns. Attempts to stain the isoenzyme patterns by the “lead conversion” methods gave very poor results. Depnrtntent of Child Health, Kirg’s College Hospital, Lo&on, S. E.5 (U.K.)
J. R. RAWSTRON S. H. NG
I S. POSEN,Ii. C. NEALE AND,J. S. CLUBB. Ann. Internal Med., 62 (1965) 1234. 2 M. HORNE, C. J. CORNISH AND S. POSEN, J. Lab. Clin. Med., 72 (1968) 905. 3 W. H. FISHMAN AND N. K. GHOSH, Advan. Clin. Chem., IO (1967) 255. 4 N. H. KORNER, J. C&n. Pathol, 15 (1962) 195. 5 bl. A. NEWTON, @art J. Med., 36 (1967) 17. 0 R. J. WIEME, Agar Grl Eelectrophoresis, Elsevier, Amsterdam, 1965, pp. 68-76, 163-164 7 J. M. YONG, J. Clin. Pathol, zo (1967) 647. 8 J. M. ALLEN AND G. J. HYNICK, J. Histochem. Cytochem., II (1963) 169. g M. M. KAPLAN AND L. ROGERS, Lancet, ii (1969) Iozg. IO E. J. KING, Micro-analysis in Medical Biochemistry, Churchill, London, 1951. p. 72.
Received Clin. Chint.
October Acta,
19, 1970
32 (197’)
303-304