- Email: [email protected]
[19] Uric acid: Functions and determination
162
FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
diethyl ether. Lutein and zeaxanthin were characterized by their spectral properties, failure to display hypsochromic shifts upon addition of ethanolic HC1, and by their relative polarity values in 80% aqueous methanol/ petroleum ether. These five xanthophylls were separated in 11 min using an isocratic solvent of acetonitrile-methanol (85 : 15). To elute the hydrocarbon fraction, a less polar solvent is necessary in rp-HPLC, and, at 11 min, the solvent was changed to hexane-methanol (25:75) and this resulted in the elution offl-carotene at approximately 17 min. We have used this system for characterizing the effluents from column chromatography, resulting in a very rapid characterization and determination of purity, based on retention times. Under circumstances of detecting column effluents, the amounts required can be scaled down by a factor of 10, permitting the analysis of nanogram quantities of carotenoid pigments. Conclusions We have presented the general principles whereby carotenoid pigments can be extracted, separated, identified, and quantitated using both TLC and rp-HPLC. The latter system, in conjunction with a variety of tests carried out on the individual fractions obtained from chromatograph effluents, results in the rapid characterization and quantitation of a variety of carotenoids, particularly those from plant tissues. Similar systems are applicable to animal systems as well as in vitro systems to which carotenoids or tissue extracts have been added.
[19] U r i c Acid: F u n c t i o n s a n d D e t e r m i n a t i o n By
PAUL HOCHSTEIN, LINDA HATCH,
and
ALEX SEVANIAN
Functions for Urate in Biological Systems Uric acid has been known for almost 100 years as an end product of purine metabolism. However, it has only been in recent years that its capacity to act as a free-radical scavenger and a potentially important biological antioxidant has been recognized. It should be noted that many hydroxylated compounds, including purine bases and their derivatives, may act as scavengers of singlet oxygen, superoxide anions, and hydroxyl radicals. Uric acid may be unique in this regard because of both its high reactivity with such chemical species and its high concentration in biologMETHODS IN ENZYMOLOGY,VOL. 105
Copyright © 1984by Academic Press, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-182005-X
[19]
URIC ACtD
163
ical fluids. It has been proposed that these attributes predict an important role for urate in oxidant and radical-induced aging and cancer. Urate may have yet additional functions, unrelated to its radical scavenging and consequent oxidation to ailantoin. Recent experiments indicate that it forms complexes with iron. 2 This reaction results in the inhibition of chelated as well as free iron-dependent oxidation of ascorbic acid and the iron-dependent peroxidation of lipids in the presence of hydroperoxides. This latter inhibition of a Fenton-type reaction, without the apparent oxidation of urate, may serve to block the formation of free radicals and the initiation of damaging reactions in membranes as well as to other biological constituents. The finding that urate may complex with iron is supportive of earlier findings of Albert 3 and has implications for the suggestions by Mazur and his colleagues that xanthine oxidase activity may function in the mobilization of iron from ferritin. 4 Finally, it has also been demonstrated that urate may regulate the synthesis of prostaglandins. 5'6 This effect is presumably mediated through its activity in scavenging hydroxyl radicals and may provide for additional roles of urate in a variety of normal and pathophysiological states. Formation of Urate The high concentration, up to 450 ~M, of urate in the plasma of man and certain primates is a consequence of the evolutionary loss of uricase (urate oxidase) and the development of efficient reabsorption mechanisms in the kidney. In mammalian tissues, free purine bases derived from nucleoside cleavage are ultimately oxidized by xanthine oxidase to yield uric acid. Purines are also derived from dietary sources and plasma levels of urate may be substantially increased in individuals on a high purine diet. Xanthine oxidase activity is present in liver and small intestinal mucosa although traces of activity have been noted in heart and skeletal muscle as well as kidney and spleen. 7 In this connection, it is important to note that xanthine oxidase activity has been found to result from the B. N. Ames, R. Cathcart, E. Schwiers,and P. Hochstein,Proc. 78, 6858 (1981).
Natl. Acad. Sci. U.S.A.
2 K. J. A. Davies, A. Sevanian, S. F. Muakkassah-Kelly, and P. Hoehstein, to be published, 1984. 3 A. Albert, Biochem. J. 54, 646 (1953). A. Mazur and A. Carlton, Blood 26, 317 (1965). s N. Ogino, S. Yamamoto, O. Hayaishi, and T. Tokuyama, Biochem. Biophys. Res. Commun. 87, 184 (1979). 6 C. Deby, G. Deby-Dupont, F. X. Noel, and L. Lavergue, Biochem. Pharmacol. 30, 2243 (1981). R. W. E. Watts, J. E. M. Watts, and J. E. Seegmiller, J. Lab. Clin. Med. 666, 688 (1965).
164
FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
modification of native xanthine dehydrogenase by limited proteolysis 8 or by the oxidation of sulfhydryl groups. 9 It seems likely that the conversion of xanthine dehydrogenase, which utilizes NAD ÷, to xanthine oxidase, which utilizes 02, may have important physiological consequences. At the present time, although uric acid formation seems to be predominantly a hepatic process, the importance of its formation in other tissues should not be minimized. It is of interest that uric acid ribonucleoside has been reported to be present in beef erythrocytes as well as in liver j° and that an antioxidant function for ribosyluric acid has been described. ~ Urate is present in the plasma, at pH 7.4, as a sodium salt (uric acid has a pK of 5.8). A small portion of uric acid, less than 5%, may be bound to plasma proteins. Its concentration in males approaches its maximum solubility (about 7 mg/100 ml) and individuals with higher levels are susceptible to gout ~2 if not increased intelligence. ~3 In a normal male about 500 mg of urate per day is excreted from a pool of about 1200 mg. The table lists the plasma concentrations of urate and some other substances with antioxidant activities. Determination of Urate In the past, the most commonly used methods for the determination of urate utilized its capacity to act as a reducing agent.~4 However, these colorimetric methods based, for example, on the reduction of sodium tungstate have inherent difficulties. These are related to the formation of turbidity, nonlinearity, and, most important, the interference by other reducing substances, e.g., glutathione. Uric acid has a characteristic absorption spectrum with a maximum at 292 nm and a molar extinction coefficient of 12,500 cm2/moi at pH 9.4. In the presence of uricase, urate is converted to allantoin which has no absorption at this wavelength. The decrease in optical density at 292 nm is a direct measure of the amount of uric acid consumed in samples.~5 This method avoids problems associated with the precipitation of protein and, 8 M. G. BaUelli, E. Della Corte, and F. Stripe, Bioehem. J. 126, 747 (1972). 9 E. Della Corte and F. Stripe, Bioehem. J. 126, 739 (1972). l0 R. C. Smith and C. M. Stricker, J. Anita. Sei. 41, 1674 (1975). H R. C. Smith, Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 1287 (1982). 12 j. B. Wyngaarden and W. N. Kelly, in "Metabolic Basis of Inherited Disease" (J. B. Stanbury, J. B. Wyngaarten, and D. S. Fredrickson, eds.). McGraw-Hill, New York, 1978. " K. S. Park, E. Inouye, and A. Asaka, £pn. J. Hum. Genet. 25, 193 (1980). 14 O. Folin, J. Biol. Chem. 101, 111 (1933). is E. Praetorius, Seand. J. Clin. Lab. Invest. 1, 222 (1949).
[19]
omc ACiD
165
PLASMA CONCENTRATIONS OF URATE AND OTHER ANTIOXIDANTSa Substance Urate Males Premenopausal females Ascorbate Normal adult Normal adult + 2 g supplement Vitamin E Carotenoids
mg/100 ml
t~M
2.6-7.5 2.0-5.7
160-450 120-340
0.7-2.5 1.7-2.8 0.5-1.6 0.09-0.12
40-140 100-160 10-40 2
" From Ref. I.
providing that appropriate blanks are utilized to correct for reactions due to other substances, is sensitive and accurate. Since the conversion of uric acid to allantoin by uricase involves the formation of H202, the reaction may be followed by coupling peroxide formation to the oxidation of o-dianisidine t6 or to the oxidation of scopoletin, t7 These methods combine the specificity of uricase activity with the ease of colorimetric or fluorometric detection. In recent years rapid and sensitive methods for the determination of urate by high-pressure liquid chromatography (HPLC) have been used.18 We have developed an HPLC method by which a variety of purines and their metabolites may be measured in biological samples or in a number of buffer systems. For example, when urate measurements in serum or urine are required, a 500-/xl aliquot is applied to an aminopropyl-NH2,500 mg Bond Elute column (Analytichem International, Harbor City, California) previously conditioned with 1.0 ml acetonitrile. Vacuum-assisted elution of the sample fluid is followed with a 500 p.l rinse of the column with acetonitrile. These eluents are discarded. Collection of a final 1.0 ml wash with 0. I M NaH2PO4 permits quantitative recovery of uric acid and ascorbic acid as well as a number of related compounds. Aqueous samples or those prepared as above are injected into a 4.6 mm x 30 cm, tzBondapak-NH2 column (Waters Assoc., Milford, Massat6 G. F. Domack and H. H. Schlicke, Ann. Biochem. 2Z, 219 (1968). 17 p. L. BIoch and G. F. Lata, Ann. Biochem. 38, I (1970). is E. J. Weinman, D. Steplock, S. C. Sansom, T. F. Knight, and H. O. Senekjian, Kidney Int. 19, 83 (1981).
166
FORMATION OR REMOVAL OF OXYGEN RADICALS
25.00
[19]
'
PUI01
2
A
4
!/
I!1
5 ,
it"~
i i 3~0
510
/
~'..
710
910
11'.0
13'.0
(mini FZG. 1. Chromatographic tracing of uric acid, ascorbic acid, and other purine metabolites. Samples were prepared in 10 mM Tris buffer, pH 7.4, at the following concentrations: (1) caffeine, 2.28 mM-2.38 min; (2) xanthine, 1.26 mM-3.40 rain; (3) inosine, 0.63 mM-4.24 min; (4) urate, !.39 mM--6.07 min; (5) ascorbate, 1.26 mM-8.09 rain. High-pressure chromatography was performed using a Perkin-Elmer series 4 instrument and an LC-85 detector. The relative absorbances at 248 nm are recorded in millivolts where 0.04 absorbance units full-scale is equivalent to 10 mV. Elution conditions are described in the text. To (using hexane) was found to be 2.07 min.
chusetts) which is eluted with 75 : 25 acetonitrile : NaH2PO4 (0.04 M) at a flow rate of 1.5 ml/min. Urate, ascorbate, and a number of other purines are detected in the eluent by spectrophotometric measurement at 248 nm. A typical chromatogram for a sample containing a mixture of ascorbic acid, uric acid and related purine metabolites is shown in Fig. 1. This mixture was prepared in 0.01 M Tris-HCl, pH 7.0, containing the following compounds: 1.26 mM ascorbate, 1.26 mM xanthine, 0.63 mM inosine, 2.28 mM caffeine, and 1.39 mM urate. A recovery of > 95% of all components was obtained when the mixture was applied to the Bond Elute preparative column described above. Interfering compounds which can be a potential problem in analyzing biological specimens include: theophylline, recovery time (rt) = 2.6 min; tyrosine, rt = 2.2 min; and dopamine, rt = 2.4 min or related metabolites.
FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
diethyl ether. Lutein and zeaxanthin were characterized by their spectral properties, failure to display hypsochromic shifts upon addition of ethanolic HC1, and by their relative polarity values in 80% aqueous methanol/ petroleum ether. These five xanthophylls were separated in 11 min using an isocratic solvent of acetonitrile-methanol (85 : 15). To elute the hydrocarbon fraction, a less polar solvent is necessary in rp-HPLC, and, at 11 min, the solvent was changed to hexane-methanol (25:75) and this resulted in the elution offl-carotene at approximately 17 min. We have used this system for characterizing the effluents from column chromatography, resulting in a very rapid characterization and determination of purity, based on retention times. Under circumstances of detecting column effluents, the amounts required can be scaled down by a factor of 10, permitting the analysis of nanogram quantities of carotenoid pigments. Conclusions We have presented the general principles whereby carotenoid pigments can be extracted, separated, identified, and quantitated using both TLC and rp-HPLC. The latter system, in conjunction with a variety of tests carried out on the individual fractions obtained from chromatograph effluents, results in the rapid characterization and quantitation of a variety of carotenoids, particularly those from plant tissues. Similar systems are applicable to animal systems as well as in vitro systems to which carotenoids or tissue extracts have been added.
[19] U r i c Acid: F u n c t i o n s a n d D e t e r m i n a t i o n By
PAUL HOCHSTEIN, LINDA HATCH,
and
ALEX SEVANIAN
Functions for Urate in Biological Systems Uric acid has been known for almost 100 years as an end product of purine metabolism. However, it has only been in recent years that its capacity to act as a free-radical scavenger and a potentially important biological antioxidant has been recognized. It should be noted that many hydroxylated compounds, including purine bases and their derivatives, may act as scavengers of singlet oxygen, superoxide anions, and hydroxyl radicals. Uric acid may be unique in this regard because of both its high reactivity with such chemical species and its high concentration in biologMETHODS IN ENZYMOLOGY,VOL. 105
Copyright © 1984by Academic Press, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-182005-X
[19]
URIC ACtD
163
ical fluids. It has been proposed that these attributes predict an important role for urate in oxidant and radical-induced aging and cancer. Urate may have yet additional functions, unrelated to its radical scavenging and consequent oxidation to ailantoin. Recent experiments indicate that it forms complexes with iron. 2 This reaction results in the inhibition of chelated as well as free iron-dependent oxidation of ascorbic acid and the iron-dependent peroxidation of lipids in the presence of hydroperoxides. This latter inhibition of a Fenton-type reaction, without the apparent oxidation of urate, may serve to block the formation of free radicals and the initiation of damaging reactions in membranes as well as to other biological constituents. The finding that urate may complex with iron is supportive of earlier findings of Albert 3 and has implications for the suggestions by Mazur and his colleagues that xanthine oxidase activity may function in the mobilization of iron from ferritin. 4 Finally, it has also been demonstrated that urate may regulate the synthesis of prostaglandins. 5'6 This effect is presumably mediated through its activity in scavenging hydroxyl radicals and may provide for additional roles of urate in a variety of normal and pathophysiological states. Formation of Urate The high concentration, up to 450 ~M, of urate in the plasma of man and certain primates is a consequence of the evolutionary loss of uricase (urate oxidase) and the development of efficient reabsorption mechanisms in the kidney. In mammalian tissues, free purine bases derived from nucleoside cleavage are ultimately oxidized by xanthine oxidase to yield uric acid. Purines are also derived from dietary sources and plasma levels of urate may be substantially increased in individuals on a high purine diet. Xanthine oxidase activity is present in liver and small intestinal mucosa although traces of activity have been noted in heart and skeletal muscle as well as kidney and spleen. 7 In this connection, it is important to note that xanthine oxidase activity has been found to result from the B. N. Ames, R. Cathcart, E. Schwiers,and P. Hochstein,Proc. 78, 6858 (1981).
Natl. Acad. Sci. U.S.A.
2 K. J. A. Davies, A. Sevanian, S. F. Muakkassah-Kelly, and P. Hoehstein, to be published, 1984. 3 A. Albert, Biochem. J. 54, 646 (1953). A. Mazur and A. Carlton, Blood 26, 317 (1965). s N. Ogino, S. Yamamoto, O. Hayaishi, and T. Tokuyama, Biochem. Biophys. Res. Commun. 87, 184 (1979). 6 C. Deby, G. Deby-Dupont, F. X. Noel, and L. Lavergue, Biochem. Pharmacol. 30, 2243 (1981). R. W. E. Watts, J. E. M. Watts, and J. E. Seegmiller, J. Lab. Clin. Med. 666, 688 (1965).
164
FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
modification of native xanthine dehydrogenase by limited proteolysis 8 or by the oxidation of sulfhydryl groups. 9 It seems likely that the conversion of xanthine dehydrogenase, which utilizes NAD ÷, to xanthine oxidase, which utilizes 02, may have important physiological consequences. At the present time, although uric acid formation seems to be predominantly a hepatic process, the importance of its formation in other tissues should not be minimized. It is of interest that uric acid ribonucleoside has been reported to be present in beef erythrocytes as well as in liver j° and that an antioxidant function for ribosyluric acid has been described. ~ Urate is present in the plasma, at pH 7.4, as a sodium salt (uric acid has a pK of 5.8). A small portion of uric acid, less than 5%, may be bound to plasma proteins. Its concentration in males approaches its maximum solubility (about 7 mg/100 ml) and individuals with higher levels are susceptible to gout ~2 if not increased intelligence. ~3 In a normal male about 500 mg of urate per day is excreted from a pool of about 1200 mg. The table lists the plasma concentrations of urate and some other substances with antioxidant activities. Determination of Urate In the past, the most commonly used methods for the determination of urate utilized its capacity to act as a reducing agent.~4 However, these colorimetric methods based, for example, on the reduction of sodium tungstate have inherent difficulties. These are related to the formation of turbidity, nonlinearity, and, most important, the interference by other reducing substances, e.g., glutathione. Uric acid has a characteristic absorption spectrum with a maximum at 292 nm and a molar extinction coefficient of 12,500 cm2/moi at pH 9.4. In the presence of uricase, urate is converted to allantoin which has no absorption at this wavelength. The decrease in optical density at 292 nm is a direct measure of the amount of uric acid consumed in samples.~5 This method avoids problems associated with the precipitation of protein and, 8 M. G. BaUelli, E. Della Corte, and F. Stripe, Bioehem. J. 126, 747 (1972). 9 E. Della Corte and F. Stripe, Bioehem. J. 126, 739 (1972). l0 R. C. Smith and C. M. Stricker, J. Anita. Sei. 41, 1674 (1975). H R. C. Smith, Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 1287 (1982). 12 j. B. Wyngaarden and W. N. Kelly, in "Metabolic Basis of Inherited Disease" (J. B. Stanbury, J. B. Wyngaarten, and D. S. Fredrickson, eds.). McGraw-Hill, New York, 1978. " K. S. Park, E. Inouye, and A. Asaka, £pn. J. Hum. Genet. 25, 193 (1980). 14 O. Folin, J. Biol. Chem. 101, 111 (1933). is E. Praetorius, Seand. J. Clin. Lab. Invest. 1, 222 (1949).
[19]
omc ACiD
165
PLASMA CONCENTRATIONS OF URATE AND OTHER ANTIOXIDANTSa Substance Urate Males Premenopausal females Ascorbate Normal adult Normal adult + 2 g supplement Vitamin E Carotenoids
mg/100 ml
t~M
2.6-7.5 2.0-5.7
160-450 120-340
0.7-2.5 1.7-2.8 0.5-1.6 0.09-0.12
40-140 100-160 10-40 2
" From Ref. I.
providing that appropriate blanks are utilized to correct for reactions due to other substances, is sensitive and accurate. Since the conversion of uric acid to allantoin by uricase involves the formation of H202, the reaction may be followed by coupling peroxide formation to the oxidation of o-dianisidine t6 or to the oxidation of scopoletin, t7 These methods combine the specificity of uricase activity with the ease of colorimetric or fluorometric detection. In recent years rapid and sensitive methods for the determination of urate by high-pressure liquid chromatography (HPLC) have been used.18 We have developed an HPLC method by which a variety of purines and their metabolites may be measured in biological samples or in a number of buffer systems. For example, when urate measurements in serum or urine are required, a 500-/xl aliquot is applied to an aminopropyl-NH2,500 mg Bond Elute column (Analytichem International, Harbor City, California) previously conditioned with 1.0 ml acetonitrile. Vacuum-assisted elution of the sample fluid is followed with a 500 p.l rinse of the column with acetonitrile. These eluents are discarded. Collection of a final 1.0 ml wash with 0. I M NaH2PO4 permits quantitative recovery of uric acid and ascorbic acid as well as a number of related compounds. Aqueous samples or those prepared as above are injected into a 4.6 mm x 30 cm, tzBondapak-NH2 column (Waters Assoc., Milford, Massat6 G. F. Domack and H. H. Schlicke, Ann. Biochem. 2Z, 219 (1968). 17 p. L. BIoch and G. F. Lata, Ann. Biochem. 38, I (1970). is E. J. Weinman, D. Steplock, S. C. Sansom, T. F. Knight, and H. O. Senekjian, Kidney Int. 19, 83 (1981).
166
FORMATION OR REMOVAL OF OXYGEN RADICALS
25.00
[19]
'
PUI01
2
A
4
!/
I!1
5 ,
it"~
i i 3~0
510
/
~'..
710
910
11'.0
13'.0
(mini FZG. 1. Chromatographic tracing of uric acid, ascorbic acid, and other purine metabolites. Samples were prepared in 10 mM Tris buffer, pH 7.4, at the following concentrations: (1) caffeine, 2.28 mM-2.38 min; (2) xanthine, 1.26 mM-3.40 rain; (3) inosine, 0.63 mM-4.24 min; (4) urate, !.39 mM--6.07 min; (5) ascorbate, 1.26 mM-8.09 rain. High-pressure chromatography was performed using a Perkin-Elmer series 4 instrument and an LC-85 detector. The relative absorbances at 248 nm are recorded in millivolts where 0.04 absorbance units full-scale is equivalent to 10 mV. Elution conditions are described in the text. To (using hexane) was found to be 2.07 min.
chusetts) which is eluted with 75 : 25 acetonitrile : NaH2PO4 (0.04 M) at a flow rate of 1.5 ml/min. Urate, ascorbate, and a number of other purines are detected in the eluent by spectrophotometric measurement at 248 nm. A typical chromatogram for a sample containing a mixture of ascorbic acid, uric acid and related purine metabolites is shown in Fig. 1. This mixture was prepared in 0.01 M Tris-HCl, pH 7.0, containing the following compounds: 1.26 mM ascorbate, 1.26 mM xanthine, 0.63 mM inosine, 2.28 mM caffeine, and 1.39 mM urate. A recovery of > 95% of all components was obtained when the mixture was applied to the Bond Elute preparative column described above. Interfering compounds which can be a potential problem in analyzing biological specimens include: theophylline, recovery time (rt) = 2.6 min; tyrosine, rt = 2.2 min; and dopamine, rt = 2.4 min or related metabolites.