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Chemical instability of [2-14C]uric acid in alkaline solution: The effect on observed kinetics of urate transport in human erythrocytes
ANALYTICAL
BIOCHEMISTRY
75,640-645
(1976)
Chemical Instability of [2J%]lJric Acid in Alkaline Solution: The Effect on Observed Kinetics of Urate Transport in Human Erythrocytes [2-“C]Uric acid. in strongly alkaline solution, is chemically unstable when stored at +4”C or -20°C. Two major degradation products occur, which cochromatograph with allantoin and allantoic acid. The nonenzymic decomposition of [2-‘T]uric acid markedly alters the observed kinetics of uric acid efflux from human erythrocytes.
Studies of the transport of uric acid in biological systems, both in riro and in vitro. involve use of alkaline solutions. This is due to poor solubility of the free acid form of the urate molecule at neutral pH (1). In tissues lacking uricase, the oxidative decomposition of uric acid by peroxidising haemoproteins at physiological pH, has been well characterised (2,3). By contrast, although there are indications that aqueous solutions of uric acid are unstable (4,.5), little attention has been paid to the nature and extent of nonenzymic uricolysis in such solutions. During a study of uric acid transport across red blood cell membranes, we have made use of [2-14C]uric acid obtained from The Radiochemical Centre, Amersham, England. This preparation is stated to be 98% pure chromatographically (data sheet CFA 221) and has served as a source material for studies of urate transport by various workers in different parts of the world (5-7). Quantitative discrepancies which appeared in some kinetic parameters of the transport system we were investigating led us to examine, chromatographically, samples of [2-W]uric acid which had been stored in aqueous solution at -2o”C, for varying periods of time. On finding substantial amounts of radioactive impurities, we proceeded to study the effect of storage of [2-lAC]uric acid on the observed kinetic behaviour of the transport system in human erythrocyte membranes. Variations in chemical environment, pH, and temperature of storage were assessed for their influence on the stability of [2-ldC]uric acid in solution. Samples of [2-l”C]uric acid (50 &i), prepared from the same batch, were dissolved in 1 ml of 100 mM NaOH, 1 mM NaOH, or 100 mM Na,HPO,. Each sample was further divided into two aliquots which were stored at 4°C and -20°C. The [2J4C]uric acid solutions were used immediately, and also after varying lengths of storage, for the determination of rate constants for [2-14C]uric acid efflux from preloaded red blood cells (8). Freshly drawn heparinised venous blood from healthy volunteers was spun down at 3000g. The plasma and buffy layer were 640 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
TABLE THE
EFFECT
OF STORAGE
OF [2-Y]URIC
TEMPERATURES, ON THE MEASURED 0.25 mu [Y]URIC ACID FROM
Efflux rate constant for [2-‘Y]uric acid on day of prepardtion (min-‘) Solvent
+”
-
100 mM NaOH, pH 13.0
0.073 1
0.0102
1 mM NaOH, pH 10.0
0.0893
100 mM Na,HPO, pH 9.5
0.0754
n The b (+),
641
COMMUNICATIONS I
ACID
IN VARIOUS
SOLVENTS,
RATE CONSTANT PRELOADED RED
Percentage constant Temperature of storage (“C)
Day
details of media In the presence
1. (-).
6
Day
34
+
-
4
59.4 39.5
93.1 70.6
18.9 11.8
51.0 81.9
4
115.6 92.9
97.2 87.2
62.8 62.3
79.2 73.1
4
101.6 110.9
103.0 107.8
82.2 85.1
82.6 X7.8
-20 are given in Fig. of I mM adenine:
of measured rate relative to day of preparation
-
-20 0.0102
OF
+
-20 0.0109
AT DIFFERING
FOR EFFLUX BLOOD CELLS”
without
added
adenine.
discarded and the packed red blood cells were washed three times with ice-cold Tris-NaCl medium containing 0.25 mM uric acid. Appearance of radioactivity in the medium, from cells previously loaded with [2-IV]uric acid (0.4 ,&il~mol), was measured and the fractional rate of decrease of the cell/medium ratio of radioactivity calculated. These experiments were performed in the presence or absence of 1 mM hypoxanthine or adenine, inhibitors of uric acid transport (9,lO). in order to determine the contribution of a purine-sensitive pathway to the overall efflux of urate. Studies were repeated. using red cells of the same healthy subject for each series, after prolonged periods of storage of the labelled uric acid. A l-p1 portion of each radioactive solution was also removed on the day of each experiment and subjected to paper chromatography to identify the products of any degradation occurring during storage. The kinetic analysis appears in Table 1 and shows that none of the solvents used completely stabilises [2-1JC]uric acid, although the rate of uricolysis is less in both 1 mM NaOH and 100 mM Na,HPO, than in 100 mM NaOH. Thus, for example, the efflux rate constant after 34 days of storage in 100 mM NaOH fell to 1% of its initial value, whereas the rate constants obtained after similar storage in 1 mM NaOH and 100 mM N&HPO, were 63% and 82% of initial values, respectively. In all of the solvent systems used the rate constant, in the presence of adenine, did not fall by more than 30%.
642
SHORT COMMUNICATIONS
FIG. 1. Effect of storage of solutions of [2-Y]uric acid at -20°C on the observed rate of isotopic efflux from preloaded red blood cells. Washed human erythrocytes, from a healthy volunteer, were preloaded in a medium containing NaCl, 150 mM; Tris-HCI, pH 7.4, 10 mM; [2-*4C]uric acid, 0.25 mM (0.4 &i/qol). Cells were resuspended (haematoctit, 10%) in nonradioactive medium, and the time course of the appearance of radioactivity in the medium was followed. The fraction of the initial intracellular radioactivity that remained inside the cells at each time was calculated and plotted on a logarithmic scale against time. Rate constants were obtained from the slopes of the straight lines obtained. (0), Control medium; (O), + 1 mM adenine.
A second study was undertaken with 100 mrvr NaOH as the storage medium, using blood from the same subject. The value for the efflux rate constant, in the absence of inhibitor, dropped from 0.061 to 0.037 min-l (3% fall) upon storage of the radioactive uric acid for only 3 days (Fig. 1). Further storage resulted in an exponential decline in the measured rate constant to a value of 24% of control after 67 days of storage. This represents a fractional rate of decrease of 0.023 day-‘. Again, there was a smaller drop in the measured rate constant in the presence of the transport inhibitor. The initial value of 0.007 min-’ did not fall by more than 25% over the period of the study. These results would be expected if simple diffusion accounted for most of the observed efflux in the presence of inhibitor. In the system used, the total amount of radioactivity inside the cells at zero time was independent of length of storage of the [2-*4C]uric acid. Since the gradient of radioactivity across the membrane rather than the chemical nature of the transported material is the factor controlling efflux by diffusion, little effect on the rate constant for efflux would have been anticipated. Examination of the chromatographs and their accompanying radioscans revealed significant changes in the chemical characteristics of the radioactive solution with the passage of time (Fig. 2). Initially, most of the radioactivity appeared in the uric acid peak, with only a small residual fraction which cochromatographed with allantoin. The nature and extent of such impurity, in fresh solutions of [2J4C]uric acid, has been reported
SHORT
COMMUNICATIONS
643
FIG. 2. Chromatographs with matching radioscans of solutions of [2-‘*C]uric acid. Aliquots (1 ~1) of the radioactive solutions were applied to chromatography paper and run in a solvent system containing n-butanol:pyridine:water (I: 1: I). Marker solutions of unlabelled uric acid, allantoin, allantoic acid, and hypoxanthine were also run. Spots were identified under ultraviolet light, and stained with Ehrlich’s reagent (2% p-dimethylaminobenzaldehyde in 20% HCI). and the radioactive distribution was measured with a Tracerlab 4~ chromatogram scanner. The upper tracings were obtained with freshly prepared solutions of [2-Y]uric acid. Remaining tracings from above downwards were obtained using solutions stored for: 3. 39. and 67 days, respectively. Abbreviations: II, uric acid: a, allantoin: h, hypoxanthine: aa. allantoic acid.
similarly by other workers (11,12). With continued storage, there was a progressive increase in the amount of radioactivity found in the allantoin peak. After 1 month, a third fraction appeared which ran just behind uric acid in the solvent system used. This fraction was found to correspond with allantoic acid. No radioactivity cochromatographed with hypoxanthine. When 1 mM NaOH or 100 mM Na,HPO, was used to dissolve the [2-14C]uric acid, similar chromatographs were obtained, but the increased stability of the uric acid was reflected in delayed
644
SHORT COMMUNICATIONS
appearance of degradation products to an extent which corresponded well with results of the kinetic analysis. The products of uric acid breakdown in aqueous solution appear to be allantoin and allantoic acid, which are the main products of urate oxidation both in vitro (12) and in viva (13). The degradation could not have been due to bacterial contamination, since a sample of [2J4C]uric acid, stored in 100 mM NaOH + 1% chloroform, displayed a similar chromatographic behaviour. as did the sample stored in 100 mM NaOH alone. These findings indicate that aqueous, strongly alkaline solutions of [2-llC]uric acid are unstable, even when stored at -20°C. In a number of publications, chromatographic purity of uric acid has been tested either before or immediately after experiments (4.5,14), and a purity ranging from 80 to 98% has been reported. It has also been stated that aqueous solutions of [2-1”C]uric acid, in NaHCO,, are chromatographitally stable (6). Unfortunately, none of these studies gives an indication of exactly how long the uric acid had been stored in solution before use. The conclusions of the present study have a bearing on the design and interpretation of experiments where small quantities of solutions of radioactive urate are used in vitro, leaving more to be stored for further use. Unless the solutions are discarded after a few days of storage and a quantitative chemical analysis of the radioactive solutions is undertaken routinely, variations in the specific radioactivity of the [2-14C]uric acid may go undetected. It is possible that some of the allantoin and allantoic acid found in human urine (13) is derived not only from the metabolic activity of the intestinal flora (15) but also from nonenzymic uricolysis described above. If this were so, chemical characterisation of the decomposition products might help to explain observed discrepancies between the in rive metabolism of [14C]uric acid and [15N]uric acid (15). REFERENCES 1. Varley. J. (1966) Practical Clinical Biochemistry, 3rd ed. pp. 157- 162, Heinemann, London. 2. Canellakis, E. S., Tuttle, A. L.. and Cohen, P. P. (1955) J. Biol. Chem. 213,397-404. 3. Howell, R. R., and Wyngaarden. J. B. (l%O) J. Biol. Chem. 235, 3544-3550. 4. Lassen, U. V. (1961) Biochim. Biophys. Acra 53, 557-569. 5. Abramson. R. G.. Levitt, M. F., Maesaka, J. K.. and Katz. J. H. (1974) J. Appl. Physiol.
6. 7. 8. 9. 10. Il. 12. 13.
36, 500-505.
Kramp. R. A., and Lenoir, R. (1975) Amer. J. Physiol. 228, 875-883. Greger, R., Lang, F., Puls, F., and Deetjen. P. (1974) Pj?i@ers Arch. 352, 121- 133. Lant, A. F. (1975) Posfgrad. Med. J. 51 (Suppl. 6). 35-42. Overgaard-Hansen, K., and Lassen. U. V. (1959) Nature (London) 184, 553-554. Christensen, H. N.. and Jones, J. C. (196115. Biol. Chem. 236, 76-80. Dantzler, W. H. (1969) Amer. J. Physiol. 217, 1510-1519. Freedman, T. B.. and Merril. C. R. (1973) Anal. Biochem. 55, 292-296. Sorensen, L. B. (1960) Stand. J. C/in. Lab. Imsest., 12 (Suppl. 54). 112-129.
SHORT COMMUNICATIONS
645
E. F. (1975) Biochim. Biophps. Acta 14. Podevin. R. A., and Boumendil-Podevin, 375, 106- 114. 15. Wyngaarden, J. B.. and Kelley. W. N. (1972) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden. J. B., and Fredrickson, D. S., eds.). 3rd ed.. pp. 889-968. McGraw-Hill, New York.
B. A. BROOKS A. F. LANT Department of Therapeutics Westminster Hospital London S WIP 2AP, England Received November 6. 1975: accepted May 20, 1976
BIOCHEMISTRY
75,640-645
(1976)
Chemical Instability of [2J%]lJric Acid in Alkaline Solution: The Effect on Observed Kinetics of Urate Transport in Human Erythrocytes [2-“C]Uric acid. in strongly alkaline solution, is chemically unstable when stored at +4”C or -20°C. Two major degradation products occur, which cochromatograph with allantoin and allantoic acid. The nonenzymic decomposition of [2-‘T]uric acid markedly alters the observed kinetics of uric acid efflux from human erythrocytes.
Studies of the transport of uric acid in biological systems, both in riro and in vitro. involve use of alkaline solutions. This is due to poor solubility of the free acid form of the urate molecule at neutral pH (1). In tissues lacking uricase, the oxidative decomposition of uric acid by peroxidising haemoproteins at physiological pH, has been well characterised (2,3). By contrast, although there are indications that aqueous solutions of uric acid are unstable (4,.5), little attention has been paid to the nature and extent of nonenzymic uricolysis in such solutions. During a study of uric acid transport across red blood cell membranes, we have made use of [2-14C]uric acid obtained from The Radiochemical Centre, Amersham, England. This preparation is stated to be 98% pure chromatographically (data sheet CFA 221) and has served as a source material for studies of urate transport by various workers in different parts of the world (5-7). Quantitative discrepancies which appeared in some kinetic parameters of the transport system we were investigating led us to examine, chromatographically, samples of [2-W]uric acid which had been stored in aqueous solution at -2o”C, for varying periods of time. On finding substantial amounts of radioactive impurities, we proceeded to study the effect of storage of [2-lAC]uric acid on the observed kinetic behaviour of the transport system in human erythrocyte membranes. Variations in chemical environment, pH, and temperature of storage were assessed for their influence on the stability of [2-ldC]uric acid in solution. Samples of [2-l”C]uric acid (50 &i), prepared from the same batch, were dissolved in 1 ml of 100 mM NaOH, 1 mM NaOH, or 100 mM Na,HPO,. Each sample was further divided into two aliquots which were stored at 4°C and -20°C. The [2J4C]uric acid solutions were used immediately, and also after varying lengths of storage, for the determination of rate constants for [2-14C]uric acid efflux from preloaded red blood cells (8). Freshly drawn heparinised venous blood from healthy volunteers was spun down at 3000g. The plasma and buffy layer were 640 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
TABLE THE
EFFECT
OF STORAGE
OF [2-Y]URIC
TEMPERATURES, ON THE MEASURED 0.25 mu [Y]URIC ACID FROM
Efflux rate constant for [2-‘Y]uric acid on day of prepardtion (min-‘) Solvent
+”
-
100 mM NaOH, pH 13.0
0.073 1
0.0102
1 mM NaOH, pH 10.0
0.0893
100 mM Na,HPO, pH 9.5
0.0754
n The b (+),
641
COMMUNICATIONS I
ACID
IN VARIOUS
SOLVENTS,
RATE CONSTANT PRELOADED RED
Percentage constant Temperature of storage (“C)
Day
details of media In the presence
1. (-).
6
Day
34
+
-
4
59.4 39.5
93.1 70.6
18.9 11.8
51.0 81.9
4
115.6 92.9
97.2 87.2
62.8 62.3
79.2 73.1
4
101.6 110.9
103.0 107.8
82.2 85.1
82.6 X7.8
-20 are given in Fig. of I mM adenine:
of measured rate relative to day of preparation
-
-20 0.0102
OF
+
-20 0.0109
AT DIFFERING
FOR EFFLUX BLOOD CELLS”
without
added
adenine.
discarded and the packed red blood cells were washed three times with ice-cold Tris-NaCl medium containing 0.25 mM uric acid. Appearance of radioactivity in the medium, from cells previously loaded with [2-IV]uric acid (0.4 ,&il~mol), was measured and the fractional rate of decrease of the cell/medium ratio of radioactivity calculated. These experiments were performed in the presence or absence of 1 mM hypoxanthine or adenine, inhibitors of uric acid transport (9,lO). in order to determine the contribution of a purine-sensitive pathway to the overall efflux of urate. Studies were repeated. using red cells of the same healthy subject for each series, after prolonged periods of storage of the labelled uric acid. A l-p1 portion of each radioactive solution was also removed on the day of each experiment and subjected to paper chromatography to identify the products of any degradation occurring during storage. The kinetic analysis appears in Table 1 and shows that none of the solvents used completely stabilises [2-1JC]uric acid, although the rate of uricolysis is less in both 1 mM NaOH and 100 mM Na,HPO, than in 100 mM NaOH. Thus, for example, the efflux rate constant after 34 days of storage in 100 mM NaOH fell to 1% of its initial value, whereas the rate constants obtained after similar storage in 1 mM NaOH and 100 mM N&HPO, were 63% and 82% of initial values, respectively. In all of the solvent systems used the rate constant, in the presence of adenine, did not fall by more than 30%.
642
SHORT COMMUNICATIONS
FIG. 1. Effect of storage of solutions of [2-Y]uric acid at -20°C on the observed rate of isotopic efflux from preloaded red blood cells. Washed human erythrocytes, from a healthy volunteer, were preloaded in a medium containing NaCl, 150 mM; Tris-HCI, pH 7.4, 10 mM; [2-*4C]uric acid, 0.25 mM (0.4 &i/qol). Cells were resuspended (haematoctit, 10%) in nonradioactive medium, and the time course of the appearance of radioactivity in the medium was followed. The fraction of the initial intracellular radioactivity that remained inside the cells at each time was calculated and plotted on a logarithmic scale against time. Rate constants were obtained from the slopes of the straight lines obtained. (0), Control medium; (O), + 1 mM adenine.
A second study was undertaken with 100 mrvr NaOH as the storage medium, using blood from the same subject. The value for the efflux rate constant, in the absence of inhibitor, dropped from 0.061 to 0.037 min-l (3% fall) upon storage of the radioactive uric acid for only 3 days (Fig. 1). Further storage resulted in an exponential decline in the measured rate constant to a value of 24% of control after 67 days of storage. This represents a fractional rate of decrease of 0.023 day-‘. Again, there was a smaller drop in the measured rate constant in the presence of the transport inhibitor. The initial value of 0.007 min-’ did not fall by more than 25% over the period of the study. These results would be expected if simple diffusion accounted for most of the observed efflux in the presence of inhibitor. In the system used, the total amount of radioactivity inside the cells at zero time was independent of length of storage of the [2-*4C]uric acid. Since the gradient of radioactivity across the membrane rather than the chemical nature of the transported material is the factor controlling efflux by diffusion, little effect on the rate constant for efflux would have been anticipated. Examination of the chromatographs and their accompanying radioscans revealed significant changes in the chemical characteristics of the radioactive solution with the passage of time (Fig. 2). Initially, most of the radioactivity appeared in the uric acid peak, with only a small residual fraction which cochromatographed with allantoin. The nature and extent of such impurity, in fresh solutions of [2J4C]uric acid, has been reported
SHORT
COMMUNICATIONS
643
FIG. 2. Chromatographs with matching radioscans of solutions of [2-‘*C]uric acid. Aliquots (1 ~1) of the radioactive solutions were applied to chromatography paper and run in a solvent system containing n-butanol:pyridine:water (I: 1: I). Marker solutions of unlabelled uric acid, allantoin, allantoic acid, and hypoxanthine were also run. Spots were identified under ultraviolet light, and stained with Ehrlich’s reagent (2% p-dimethylaminobenzaldehyde in 20% HCI). and the radioactive distribution was measured with a Tracerlab 4~ chromatogram scanner. The upper tracings were obtained with freshly prepared solutions of [2-Y]uric acid. Remaining tracings from above downwards were obtained using solutions stored for: 3. 39. and 67 days, respectively. Abbreviations: II, uric acid: a, allantoin: h, hypoxanthine: aa. allantoic acid.
similarly by other workers (11,12). With continued storage, there was a progressive increase in the amount of radioactivity found in the allantoin peak. After 1 month, a third fraction appeared which ran just behind uric acid in the solvent system used. This fraction was found to correspond with allantoic acid. No radioactivity cochromatographed with hypoxanthine. When 1 mM NaOH or 100 mM Na,HPO, was used to dissolve the [2-14C]uric acid, similar chromatographs were obtained, but the increased stability of the uric acid was reflected in delayed
644
SHORT COMMUNICATIONS
appearance of degradation products to an extent which corresponded well with results of the kinetic analysis. The products of uric acid breakdown in aqueous solution appear to be allantoin and allantoic acid, which are the main products of urate oxidation both in vitro (12) and in viva (13). The degradation could not have been due to bacterial contamination, since a sample of [2J4C]uric acid, stored in 100 mM NaOH + 1% chloroform, displayed a similar chromatographic behaviour. as did the sample stored in 100 mM NaOH alone. These findings indicate that aqueous, strongly alkaline solutions of [2-llC]uric acid are unstable, even when stored at -20°C. In a number of publications, chromatographic purity of uric acid has been tested either before or immediately after experiments (4.5,14), and a purity ranging from 80 to 98% has been reported. It has also been stated that aqueous solutions of [2-1”C]uric acid, in NaHCO,, are chromatographitally stable (6). Unfortunately, none of these studies gives an indication of exactly how long the uric acid had been stored in solution before use. The conclusions of the present study have a bearing on the design and interpretation of experiments where small quantities of solutions of radioactive urate are used in vitro, leaving more to be stored for further use. Unless the solutions are discarded after a few days of storage and a quantitative chemical analysis of the radioactive solutions is undertaken routinely, variations in the specific radioactivity of the [2-14C]uric acid may go undetected. It is possible that some of the allantoin and allantoic acid found in human urine (13) is derived not only from the metabolic activity of the intestinal flora (15) but also from nonenzymic uricolysis described above. If this were so, chemical characterisation of the decomposition products might help to explain observed discrepancies between the in rive metabolism of [14C]uric acid and [15N]uric acid (15). REFERENCES 1. Varley. J. (1966) Practical Clinical Biochemistry, 3rd ed. pp. 157- 162, Heinemann, London. 2. Canellakis, E. S., Tuttle, A. L.. and Cohen, P. P. (1955) J. Biol. Chem. 213,397-404. 3. Howell, R. R., and Wyngaarden. J. B. (l%O) J. Biol. Chem. 235, 3544-3550. 4. Lassen, U. V. (1961) Biochim. Biophys. Acra 53, 557-569. 5. Abramson. R. G.. Levitt, M. F., Maesaka, J. K.. and Katz. J. H. (1974) J. Appl. Physiol.
6. 7. 8. 9. 10. Il. 12. 13.
36, 500-505.
Kramp. R. A., and Lenoir, R. (1975) Amer. J. Physiol. 228, 875-883. Greger, R., Lang, F., Puls, F., and Deetjen. P. (1974) Pj?i@ers Arch. 352, 121- 133. Lant, A. F. (1975) Posfgrad. Med. J. 51 (Suppl. 6). 35-42. Overgaard-Hansen, K., and Lassen. U. V. (1959) Nature (London) 184, 553-554. Christensen, H. N.. and Jones, J. C. (196115. Biol. Chem. 236, 76-80. Dantzler, W. H. (1969) Amer. J. Physiol. 217, 1510-1519. Freedman, T. B.. and Merril. C. R. (1973) Anal. Biochem. 55, 292-296. Sorensen, L. B. (1960) Stand. J. C/in. Lab. Imsest., 12 (Suppl. 54). 112-129.
SHORT COMMUNICATIONS
645
E. F. (1975) Biochim. Biophps. Acta 14. Podevin. R. A., and Boumendil-Podevin, 375, 106- 114. 15. Wyngaarden, J. B.. and Kelley. W. N. (1972) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden. J. B., and Fredrickson, D. S., eds.). 3rd ed.. pp. 889-968. McGraw-Hill, New York.
B. A. BROOKS A. F. LANT Department of Therapeutics Westminster Hospital London S WIP 2AP, England Received November 6. 1975: accepted May 20, 1976