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An assay for inorganic orthophosphate in the presence of alkaloids and related drugs
BIOCHEMICAL
MEDICINE
17, 153-157
(1977)
An Assay for Inorganic Orthophosphate in the Presence of Alkaloids and Related Drugs NICHOLAS R. PRICE' AND ALAN J. WILLIAMS* Department
of Biological Lancaster
Sciences, University LAI 4YQ. England
of Lancaster,
Received August 19. 1976
INTRODUCTION There have been many reported techniques for the determination of inorganic orthophosphate, largely utilising the formation of a yellow complex between the phosphate and a molybdenum salt, and the formation of a blue chromophore on reduction of the complex. The method of Fiske and Subbarow (1) employs l-amino-Znaphthol-Csulphonic acid as the reducing agent, and this technique is still widely used. Modifications to the basic procedure have included the direct measurement of the yellow phosphomolybdate complex to avoid reduction of susceptible compounds (2) and the extraction of the phosphomolybdate complex with organic solvents to prevent interference by inorganic ions (3, 4). Atkinson et al. (5), reported that incorporation of the non-ionic detergent Cirrasol ALN-WF, formerly called Lubrol-W, into the acid ammonium molybdate reagent prevented the interference in the phosphate assay by a number of simple compounds that adversely affect the conventional methods. Our studies on the effects of certain pharmacological agents on the active transport of calcium in muscle tissue, have led us to look for a reliable and simple method of determining levels of inorganic phosphate in the presence of alkaloids and related methyl xanthines, since these compounds form a white precipitate in the presence of phosphate and acid ammonium molybdate which renders the conventional assay inoperable. ’ Grant No. GR 3/2475 from the Natural Environment Research Council is gratefully acknowledged. 2 Present address: Division of Cardiac Medicine, Cardiothoracic Institute. London.
153 Copyri@t All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN OK&2Y44
154
PKIC’E
AND
MATERIALS
WILLIAMS
AND METHODS
Reagents
The following reagents were used: A. Acid-molybdate-Cirrasol: equal volumes of 1% ammonium molybdate in 1.8 N sulphuric acid and 1% aqueous Cirrasol ALN-WF (Lubrol-W Seravac Laboratories Ltd.): B. Acid-molybdate: acidic ammonium molybdate as above but diluted with an equal volume of distilled water in place of the Cirrasol; and C. I-amino-2-naphthol-4-sulphonic acid (ANSA): 0.25% in 15% sodium metabisulphite containing sufficient 20% sodium sulphite for complete solubilisation. Caffeine, theophylline, and atropine were obtained from Sigma Chemical Co. and quinine sulphate was obtained from British Drug Houses Ltd. Procedure
The drugs were dissolved in distilled water with warming where necessary. Aliquots of 1 ml were taken and phosphate levels produced by the addition of sodium dihydrogen orthophosphate. Either acid-molybdate or acid-molybdate-Cirrasol (1 ml) was added to each sample followed by 0.5 ml of ANSA. Because of the sensitivity of the technique, 5 ml of distilled water was added to all samples and after standing at room temperature for 10 min, optical densities were measured at 660 nm. RESULTS
Figure 1 shows the relationship between OD,, nm and phosphate concentration over the range of O-10 mg%, for both the conventional acidmolybdate method and the acid-molybdate-Cirrasol method. The increased sensitivity of the Cirrasol method is evident, the top Pi concentration giving an ODsso nm of 0.75 as compared to 0.42 for the conventional method. Figure 1 also shows the results of assays carried out in the presence of the drug compounds and indicates that the sensitivity and accuracy of the method are retained under these circumstances, if Cirrasol is included in the acid-molybdate reagent. The experimental points for the corresponding assays carried out in the absence of the detergent are not shown since on addition of the acid molybdate reagent a white precipitate is formed. In the case of the less soluble compounds such as quinine and atropine this precipitate prevents any meaningful measurements at all. However, OD,, ,,,,, can be measured for the more soluble drugs such as theophylline, but in this case the OD readings showed up to 30% variation on the control calibration compared to effectively no variation when the acid-molybdate-Cirrasol reagent was used. Table I shows the reproducibility of the Cirrasol technique. The variation from the control figures is very small. not exceeding 0.007 OD, and
PHOSPHATE
IN PRESENCE
1.55
OF ALKALOIDS
0.8
0.7
0.6
0.5 E c g L J, 5 n
0.4
d u k 0.3
0.2
0.1
0
I
2
3
4
5
PHOSPHATE
6
7
8
9
mg %
FIG. 1. Phosphate determinations. -0, assay using acid molybdate reagent, conassay using acid molybdate-Cirrasol reagent; 0, control; V, 2 x I o-3 M quinine trol; -, sulphate; Cl, lo-* M caffeine; & IO-* M theophylline; W, IO+ M atropine.
Control
0.083
0.165
0.315
0.715
Quinine 2 x 10m3 M Caffeine IO-’ M Theophylline IO-’ M
0.085 0.077 0.077
0.163 0. I43 0. I57
0.310 0.307 0.323
0.68 0.717 0.743
A&opine
0.085
0.175 0.005
0.320 0.004
0.730 0.007
IO-”
t- SE (N =
12)
‘l Figures
are
M
0.0016
optical
density
units;
total
from
five
treatments
this indicates that inclusion of Cirrasol has prevented interference assay by precipitation of these compounds.
of the
DISCUSSION
The estimation of inorganic orthophosphate by the production and measurement of the yellow complex formed with acid molybdate or its reduced derivative becomes problematical in the presence of a wide range of interfering compounds. The extremely low pH at which the test is conducted produces particular difficulties when the assay is applied to biological samples where extreme acidity may cause hydrolysis of labile organophosphate or precipitation of macromolecular compounds. Considerable difficulties have been encountered when dealing with nucleotide phosphates. Measurements of phosphate concentrations are often required to be made in the presence of ATP. for example, when adenosine triphosphatase activities are being monitored, and thus many modifications to the method of Fiske and Subbarow (I) have been suggested. The Lowry and Lopez method (6) utilises far milder conditions (pH 4) to minimise acid hydrolysis, but has the disadvantage that all samples must be carefully deproteinised beforehand. A modification of this method (5) using the non-ionic detergent Cirrasol ALN-WF, formerly called Lubrol-W. will tolerate moderate concentrations of a range of simple compounds including ATP, but at ATP concentrations exceeding 3mM, the development of the coloured complex is retarded and accuracy is lost. Recent techniques using Cirrasol and sodium dodecyl sulphate have improved the method by their ability to tolerate both protein and labile organophosphate contents (7). The problem of interference of the phosphate assay is not confined to these compounds. The presence of millimolar concentrations of alkaloids and methyl xanthines produces a white precipitate on mixing with acid-
PHOSPHATE
IN PRESENCE
OF ALKALOIDS
157
molybdate reagent even when the phosphate concentration is less than 10 &ml. In studies using such compounds the precipitate must be removed by centrifugation, a difficult operation since at low phosphate concentrations the precipitate is merely a heavy turbidity, or the drug must be removed before the assay by absorption onto activated charcoal (8, 9). The method reported here obviates such tedious and potentially inconsistent procedures by preventing the precipitation of these compounds. The samples over the whole range of phosphate concentrations tested were completely clear at every stage of the determination and the method can be used in ATPase inhibition tests providing that the ATP concentration does not exceed 3mM. In addition, the assay will tolerate the presence of membrane proteins up to a concentration of 150 E.Lg/ml. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.
Fiske, C. H., and Subbarow, Y., J. Biol. Gem. 66, 375 (1925). Marsh, B. B., Biochim. Biophys. Acta 32, 357 (1959). Berenblum, J., and Chain, E., Biochem. J. 32, 286 (1938). Martin, J. B., and Doty, D. M., Anal. Chem. 21, 965 (1949). Atkinson, A., Gatenby, A. D.. and Lowe, A. G., E&him. Biophys. Acm 3u), 19.5(1973). Lowry, 0. H., and Lopez, J. A., J. Biol. Chem. 162, 421 (1946). Chandra Rajan, J., and Klein, L., Anal. Biochem. 72, 407 (1976). Ells, H. A., and Faulkner, P. Arch. Inr. Pharmucodyn. CXIJ, 75 (1963). Fuchs, F., Gertz, E. W.. and Briggs, F. N., J. Gen. Physid. 6, 955 (1968).
MEDICINE
17, 153-157
(1977)
An Assay for Inorganic Orthophosphate in the Presence of Alkaloids and Related Drugs NICHOLAS R. PRICE' AND ALAN J. WILLIAMS* Department
of Biological Lancaster
Sciences, University LAI 4YQ. England
of Lancaster,
Received August 19. 1976
INTRODUCTION There have been many reported techniques for the determination of inorganic orthophosphate, largely utilising the formation of a yellow complex between the phosphate and a molybdenum salt, and the formation of a blue chromophore on reduction of the complex. The method of Fiske and Subbarow (1) employs l-amino-Znaphthol-Csulphonic acid as the reducing agent, and this technique is still widely used. Modifications to the basic procedure have included the direct measurement of the yellow phosphomolybdate complex to avoid reduction of susceptible compounds (2) and the extraction of the phosphomolybdate complex with organic solvents to prevent interference by inorganic ions (3, 4). Atkinson et al. (5), reported that incorporation of the non-ionic detergent Cirrasol ALN-WF, formerly called Lubrol-W, into the acid ammonium molybdate reagent prevented the interference in the phosphate assay by a number of simple compounds that adversely affect the conventional methods. Our studies on the effects of certain pharmacological agents on the active transport of calcium in muscle tissue, have led us to look for a reliable and simple method of determining levels of inorganic phosphate in the presence of alkaloids and related methyl xanthines, since these compounds form a white precipitate in the presence of phosphate and acid ammonium molybdate which renders the conventional assay inoperable. ’ Grant No. GR 3/2475 from the Natural Environment Research Council is gratefully acknowledged. 2 Present address: Division of Cardiac Medicine, Cardiothoracic Institute. London.
153 Copyri@t All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN OK&2Y44
154
PKIC’E
AND
MATERIALS
WILLIAMS
AND METHODS
Reagents
The following reagents were used: A. Acid-molybdate-Cirrasol: equal volumes of 1% ammonium molybdate in 1.8 N sulphuric acid and 1% aqueous Cirrasol ALN-WF (Lubrol-W Seravac Laboratories Ltd.): B. Acid-molybdate: acidic ammonium molybdate as above but diluted with an equal volume of distilled water in place of the Cirrasol; and C. I-amino-2-naphthol-4-sulphonic acid (ANSA): 0.25% in 15% sodium metabisulphite containing sufficient 20% sodium sulphite for complete solubilisation. Caffeine, theophylline, and atropine were obtained from Sigma Chemical Co. and quinine sulphate was obtained from British Drug Houses Ltd. Procedure
The drugs were dissolved in distilled water with warming where necessary. Aliquots of 1 ml were taken and phosphate levels produced by the addition of sodium dihydrogen orthophosphate. Either acid-molybdate or acid-molybdate-Cirrasol (1 ml) was added to each sample followed by 0.5 ml of ANSA. Because of the sensitivity of the technique, 5 ml of distilled water was added to all samples and after standing at room temperature for 10 min, optical densities were measured at 660 nm. RESULTS
Figure 1 shows the relationship between OD,, nm and phosphate concentration over the range of O-10 mg%, for both the conventional acidmolybdate method and the acid-molybdate-Cirrasol method. The increased sensitivity of the Cirrasol method is evident, the top Pi concentration giving an ODsso nm of 0.75 as compared to 0.42 for the conventional method. Figure 1 also shows the results of assays carried out in the presence of the drug compounds and indicates that the sensitivity and accuracy of the method are retained under these circumstances, if Cirrasol is included in the acid-molybdate reagent. The experimental points for the corresponding assays carried out in the absence of the detergent are not shown since on addition of the acid molybdate reagent a white precipitate is formed. In the case of the less soluble compounds such as quinine and atropine this precipitate prevents any meaningful measurements at all. However, OD,, ,,,,, can be measured for the more soluble drugs such as theophylline, but in this case the OD readings showed up to 30% variation on the control calibration compared to effectively no variation when the acid-molybdate-Cirrasol reagent was used. Table I shows the reproducibility of the Cirrasol technique. The variation from the control figures is very small. not exceeding 0.007 OD, and
PHOSPHATE
IN PRESENCE
1.55
OF ALKALOIDS
0.8
0.7
0.6
0.5 E c g L J, 5 n
0.4
d u k 0.3
0.2
0.1
0
I
2
3
4
5
PHOSPHATE
6
7
8
9
mg %
FIG. 1. Phosphate determinations. -0, assay using acid molybdate reagent, conassay using acid molybdate-Cirrasol reagent; 0, control; V, 2 x I o-3 M quinine trol; -, sulphate; Cl, lo-* M caffeine; & IO-* M theophylline; W, IO+ M atropine.
Control
0.083
0.165
0.315
0.715
Quinine 2 x 10m3 M Caffeine IO-’ M Theophylline IO-’ M
0.085 0.077 0.077
0.163 0. I43 0. I57
0.310 0.307 0.323
0.68 0.717 0.743
A&opine
0.085
0.175 0.005
0.320 0.004
0.730 0.007
IO-”
t- SE (N =
12)
‘l Figures
are
M
0.0016
optical
density
units;
total
from
five
treatments
this indicates that inclusion of Cirrasol has prevented interference assay by precipitation of these compounds.
of the
DISCUSSION
The estimation of inorganic orthophosphate by the production and measurement of the yellow complex formed with acid molybdate or its reduced derivative becomes problematical in the presence of a wide range of interfering compounds. The extremely low pH at which the test is conducted produces particular difficulties when the assay is applied to biological samples where extreme acidity may cause hydrolysis of labile organophosphate or precipitation of macromolecular compounds. Considerable difficulties have been encountered when dealing with nucleotide phosphates. Measurements of phosphate concentrations are often required to be made in the presence of ATP. for example, when adenosine triphosphatase activities are being monitored, and thus many modifications to the method of Fiske and Subbarow (I) have been suggested. The Lowry and Lopez method (6) utilises far milder conditions (pH 4) to minimise acid hydrolysis, but has the disadvantage that all samples must be carefully deproteinised beforehand. A modification of this method (5) using the non-ionic detergent Cirrasol ALN-WF, formerly called Lubrol-W. will tolerate moderate concentrations of a range of simple compounds including ATP, but at ATP concentrations exceeding 3mM, the development of the coloured complex is retarded and accuracy is lost. Recent techniques using Cirrasol and sodium dodecyl sulphate have improved the method by their ability to tolerate both protein and labile organophosphate contents (7). The problem of interference of the phosphate assay is not confined to these compounds. The presence of millimolar concentrations of alkaloids and methyl xanthines produces a white precipitate on mixing with acid-
PHOSPHATE
IN PRESENCE
OF ALKALOIDS
157
molybdate reagent even when the phosphate concentration is less than 10 &ml. In studies using such compounds the precipitate must be removed by centrifugation, a difficult operation since at low phosphate concentrations the precipitate is merely a heavy turbidity, or the drug must be removed before the assay by absorption onto activated charcoal (8, 9). The method reported here obviates such tedious and potentially inconsistent procedures by preventing the precipitation of these compounds. The samples over the whole range of phosphate concentrations tested were completely clear at every stage of the determination and the method can be used in ATPase inhibition tests providing that the ATP concentration does not exceed 3mM. In addition, the assay will tolerate the presence of membrane proteins up to a concentration of 150 E.Lg/ml. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.
Fiske, C. H., and Subbarow, Y., J. Biol. Gem. 66, 375 (1925). Marsh, B. B., Biochim. Biophys. Acta 32, 357 (1959). Berenblum, J., and Chain, E., Biochem. J. 32, 286 (1938). Martin, J. B., and Doty, D. M., Anal. Chem. 21, 965 (1949). Atkinson, A., Gatenby, A. D.. and Lowe, A. G., E&him. Biophys. Acm 3u), 19.5(1973). Lowry, 0. H., and Lopez, J. A., J. Biol. Chem. 162, 421 (1946). Chandra Rajan, J., and Klein, L., Anal. Biochem. 72, 407 (1976). Ells, H. A., and Faulkner, P. Arch. Inr. Pharmucodyn. CXIJ, 75 (1963). Fuchs, F., Gertz, E. W.. and Briggs, F. N., J. Gen. Physid. 6, 955 (1968).