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Fluorometric determination of xanthine and hypoxanthine in tissue
291
Clinica Chimica Acta, 95 (1979) 291-299 @ Elsevier/North-Holland Biomedical Press
CCA 1053
FLUOROMETRIC HYPOXANTHINE
TAKAHIKO
DETERMINATION IN TISSUE
OF XANTHINE
AND
SUM1 * and YURI UMEDA
Division of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-l-8, Kamikitazawa, Setagaya-Ku, Tokyo 156 (Japan) (Received
December
21st, 1978)
Summary A method was developed to determine the total content of the oxypurines, xanthine and hypoxanthine, in animal tissues. The developed method was constructed mainly from the following successive steps: (1) conversion of the oxypurines to uric acid and hydrogen peroxide by xanthine oxidase; (2) decomposition of the hydrogen peroxide by catalase and subsequent inactivation of this enzyme; (3) fluorometric measurement of the uric acid based on the coupled enzyme reaction of uricase and peroxidase. In applying this method to a sample containing uric acid, preliminary removal of this uric acid was necessary and this was carried out by treating the sample with uricase, followed by subsequent inactivation of this enzyme. The present method was more specific than the existing fluorometric method and permitted to measure the total content of the oxypurines (as low as 1 nmol) without mutual separation of them. The actual application of this method to the rat liver was demonstrated together with the method to prepare the tissue sample for the assay.
Introduction The measurement of the oxypurines, xanthine and hypoxanthine, in biological samples has been increasingly required for clinical or laboratory investigations on gout and other metabolic diseases related to puke metabolism. Spectrofluorometric [l] and spectrophotometric [ 2,3] methods by using xanthine oxidase have been developed for this purpose. Although the fluorometric method, which is principally a peroxidase-aided assay of the hydrogen peroxide produced from the oxypurines by xanthine oxidase, is simple and highly sensitive * To
whom
correspondence
should
be addressed.
292
[ 11, this method has two severe defects when applied to biological samples. The one is a lack of specificity because xanthine oxidase attacks not only xanthine and hypoxanthine but also other oxypurines, aldehydes, etc. [4], and the other one is a requirement of mutual separation of xanthine and hypoxanthine to determine a total content of their mixture because hydrogen peroxide produced is 1 mol/mol from xanthine and 2 mol/mol, from hypoxanthine. To overcome these defects, we have developed an improved fluorometric method which is constructed by connecting the conversion of the oxypurines to uric acid with the determination of the resultant uric acid by using uricase-peroxidase system [ 51. Principle The procedure of the developed method consists of the following successive steps: (1) conversion of the oxypurines to uric acid and hydrogen peroxide by xanthine oxidase, (2) destruction of the hydrogen peroxide by catalase, (3) heat-inactivation of the catalase, (4) conversion of the uric acid to allantoin and hydrogen peroxide by uricase and (5) fluorometry of the hydrogen peroxide from the uric acid by the peroxidase reaction coupled with a fluorophor, p-hydroxyphenylacetic acid; (4) with (5) has already been utilized by us to measure plasma uric acid [ 51. According to this procedure, hydrogen peroxide is produced successively from the oxypurines and the uric acid, whereas the former hydrogen peroxide. is destroyed by the catalase but the latter is no more destroyed by this enzyme because of the heat-inactivation. Therefore, the hydrogen peroxide to be estimated b,y the peroxidase system is not the one from the oxypurines themselves but the other one from their common metabolite, uric acid. As a result, one mol of both xanthine and hypoxanthine is determined finally as one mol of hydrogen peroxide, and the specificity of the production of the hydrogen peroxide to be estimated does not depend on the non-specific enzyme, xanthine oxidase, but on uricase which is highly specific for uric acid. These may promise to remove the potential defects of the existing fluorometric method. To apply this method to samples containing uric acid, a preliminary complete removal of the uric acid is necessary. In the proposed method, this is carried out by pretreating the samples with uricase, which is subsequently inactivated by heating to prevent this uricase from oxidizing the uric acid of the oxypurine-origin before the catalase treatment; the hydrogen peroxide produced by this pre-treatment is to be destroyed at the catalase step. Materials and methods Reagents Buffers. Phosphate buffer, 0.05 mol/l, pH 8.0; Tris-HCl buffer, 0.05 mol/l, pH 9.0. Xanthine oxidase solution. 100 munits of milk xanthine oxidase (0.75 units/ mg of protein) in 1.0 ml of the Tris-HCl buffer. This solution should be prepared every experiment. Catalase stock solution. 750 units of catalase (30 000 units/mg of solid) in
293
1.0 ml of 30% glycerol (with 10% ethanol). This is stable for at least 4 weeks at 4°C. Cat&se working solution. This solution (40 units/ml) is prepared by diluting the stock solution with the phosphate buffer. This should be prepared every experiment. Uricase solution. 15 munits of uricase (Type II, 15 units/g of solid) in 1.0 ml of the phosphate buffer. This is stable for at least 4 weeks in a frozen state (-20°C). Peroxidase stock solution. 200 units of horseradish peroxidase (195 units/mg of solid) in 1.0 ml of the phosphate buffer. This is preserved for at least 4 weeks in a frozen state (-20°C). p-hydroxyphenylucetic acid (pHPAA) stock solution. 4 mg of pHPAA in 1.0 ml of water. This solution is stable for at least 4 weeks in a frozen state (-20°C). pHPAA-peroxiduse-buffer solution. This is prepared by mixing 0.5 ml of the pHPAA stock solution, 0.5 ml of the peroxidase stock solution and 9.0 ml of the phosphate buffer. This is stable for at least 4 weeks in a frozen state (-20°C). Standard stock solutions. Xanthine solution, 1.0 mmol/l in the Trix-HCl 1.0 mmol/l in the Tris-HCl buffer; uric acid buffer; hypoxanthine solution, solution, 0.5 mmol/l in the phosphate buffer. These are stable for at least 4 weeks in a frozen state (-20°C). The working solutions of them are prepared by diluting the respective stock solutions with water. QAE-Sephudex A-25 formute form. QAE-Sephadex A-25 is regenerated and then converted to the formate ion. This is stored in the form of a slurry at 4°C. All the enzymes used here were purchased from Sigma Chemical Co., and QAE-Sephadex A-25 was from Pharmacia. Other chemicals were analytical grade commercial products. Appuru tus Fluorescence is measured with a Hitachi spectrofluorometer (MFP-4, Hitachi Ltd.) equipped with a xenon arc lamp. Slit widths for excitation and emission light were 5.0 and 10.0 nm, respectively. A Hitachi linear recorder was coupled with the fluorometer. Standard assay method in the absence of uric acid To 200 ~1 of oxypurine sample containing no uric acid, 80 ~1 of xanthine oxidase solution is added, and incubated for 15 min at 27°C. To the mixture, 20 ~1 of catalase solution is next added, and incubated for 20 min at 27°C. After the incubation, the mixture is heated for 10 min in a boiling-water bath to inactivate the catalase and then cooled in ice water. Next, 100 ~1 of uricase solution is added to the mixture and incubated for 30 min at 27°C. After adding 1.0 ml of pHPAA-peroxidase-buffer solution, the mixture is next incubated for 30 min at 27°C. After the incubation, the fluorescence is measured at an excitation wavelength of 317 nm and an emission wavelength of 414 nm [ 51. A sample in which xanthine oxidase solution is replaced by Tris-HCl buffer serves as a blank. The blank-fluorescence is subtracted from the sample-fluores-
294
cence, and this net increase intensity”.
will be represented
in the figures as “fluorescence
Standard assay method in the presence of uric acid To 200 ~1 of oxypurine sample containing uric acid, 100 .~l of uricase solution is added, and then incubated for 30 min at 27°C. This is next heated for 10 min in a boiling-water bath, and cooled in ice water. Then, the mixture is submitted to the oxypurine analysis; although the initial volume of the resulting oxypurine sample amounts to 300 ~1 after adding this &case treatment, the subsequent oxypurine assay is carried out according to the standard method without any modifications. Method for determining total oxypurine content in the rat liuer The rat liver extract is prepared and then submitted to the total oxypurine assay after removing interfering fluorescent substances from the extract. These preparative methods are described here. Immediately after decapitating the rat (male Wistar strain, 200-300 g body weight), the liver is removed and quickly frozen in liquid Nz. The frozen liver is homogenized with cold 0.6 mol/l HC104 (3 ml/g of tissue). The homogenate is centrifuged for 30 min at 6000 rpm, and the supernatant is served. The resultant precipitate is re-homogenized with cold 0.6 mol/l HC104 (1.5 ml/g tissue). The second homogenate is centrifuged as before, and the resultant supe~a~n~ is combined with the first. The pH of the combined extract is adjusted to 10.0 to 10.5, and the resultant precipitate is centrifuged off. l-ml aliquot from the resultarit supernatant is applied onto a QAE-Sephadex A-25 column (1.0 cm diameter, 5.0 cm long). Next, the column is washed with 12 ml of water and eluted with 12 ml of 0.05 mol/l HCOOH. The HCOOHeluent is evaporated to dryness in vacua. The resultant residue is dissolved in 1.0 ml of 0.005 mol/l NaOH. Two 0,2-ml aliquots from this solution are served each for the standard assay “in the presence of uric acid” and for the blank. Results and discussion Critical reactions in the assay procedure The critical steps in the proposed assay procedure may be the xanthine oxidase reaction, the catalase reaction and the inactivation of catalase. To check the actual progress of these reactions during the assay, the development of the fluorescence in a hypoxanthine sample (4 nmol) which was carried through the entire assay procedure was measured with varying only enzyme concentration or reaction time of the respective critical reactions. At first, the xanthine oxidase reaction was checked by varying the amount of xanthine oxidase; an exact period of the xanthine oxidase reaction is rather obscure because this enzyme is not inactivated even after the end of this reaction and may remain still active in the subsequent catalase reaction, and therefore the check was not done with respect to the t~~ou~e of xanthine oxidase reaction. As shown in Fig. 1, the fluorescence did not develop without this enzyme, increased markedly with increasing the amount of the enzyme and
295
o- 06
4 xanthine
8 12 oxidase (munits)
16
Fig. 1. Development 0:: fluorescence as a function of xanthine oxidase levels. Hypoxanthine samples (4 nmol) were carried through the entire standard assay procedure with only varying the amount of xanthine oxidase.
attained a plateau level throughout 2.0-16.0 munits of the enzyme. This indicates that the xanthine oxidase reaction actually occurred and the standard amount of xanthine oxidase was very sufficient to carry this reaction to the end under the standard assay conditions. The effect of the catalase treatment was next tested by following the change of the fluorescence as a function of the incubation time of catalase. The result (Fig. 2) shows that the fluorescence was in a maximum level without catalase treatment, then decreased with increasing the incubation time of catalase and attained a constant level throughout 15-25-min incubation. This suggests that the decreasing fluorescence was due to the hydrogen peroxide produced at the xanthine oxidase step, and the constant fluorescence reflected the other hydrogen peroxide produced at the uricase step. It should be noted that the catalase reaction went to completion within the standard 20-min incubation.
minutes Fig. 2. Development of fluorescence as a function (4 nmol) were carried through the entire standard with catalase.
of the time of catalase reaction. Hypoxanthine samples assay procedure with only varying the incubation time
296
0
2
4
6
8
10
12 minutes
Fig. 3. Development of fluorescence as a function of the time of heating for inactivating catalase. Hypoxanthine samples (4 nmol) were carried through the entire standard assay procedure with only varying the heating time for inactivating catalase.
Finally, the influence of the heat-inactivation of the catalase to the development of the fluorescence was examined by varying the heating time of the catalase in a boiling water bath. As shown in Fig. 3, the fluorescence did not develop without heating, but markedly increased by introducing this step of inactivation, the developed fluorescence being kept constant throughout 2-12 min heating. This result is consistent with a view that the catalase was capable of destroying both hydrogen peroxide produced from the oxypurine and from its metabolite, uric acid, but the latter hydrogen peroxide escaped the destruction by the preceeding heat-inactivation of this enzyme (compare with Fig. 2). In conclusion, it can be said that all of these critical reactions proceeded in the manner we had expected and almost completely ended under the standard assay conditions. Since the presented observations with these reactions were indirect, this conclusion is on the assumption that the uricase-peroxidase system was quantitatively coupled with the sequence of the critical reactions. This assumptions will be verified by the following results. Calibration curves Using the standard assay method described above, calibration curves were made with respect to the oxypurines. As shown in Fig. 4, the increase in fluorescence was proportional to concentrations of both xanthine and hypoxanthine, and these curves were completely identical with each other. This indicates that the proposed method can measure as low as 1 nmol of the oxypurines if the blank fluorescence is very low or is removed by a preliminary treatment of the sample, and that the measurement does not depend on the ratio between xanthine and hypoxanthine existing in the sample. Furthermore, the present result also gives an additional support to the view that the three critical reactions went to completion under the standard assay conditions, producing uric acid from the oxypurines and removing the hydrogen peroxide also produced from the oxypurines, thus the production of the uric acid was coupled quantitatively with the uricase-peroxidase system. It should be noted that the specificity of the present method is dependent
297
o hypoxanthine xanthine
l
oxypurines(nmols) Fig. 4. Calibration curves for xanthine and hypoxanthine. were carried through the entire standard assay procedure.
Standard
samples
of xanthine
and hypoxanthine
largely on the specificity of the uricase-peroxidase system, this system having been reported to be highly specific for uric acid [ $61 except for the interfere with ascorbic acid [6]. Preliminary
removal of contaminating
To remove uric acid adopted the preliminary sequent inactivation of inactivated by heating,
uric acid
existing very frequently in biological samples, we have treatment of the samples with uricase followed by subthis enzyme [ 71: in the present method, the uricase is which is simpler than the usual inactivation with strong
hypoxanthine(nmols)
xanthine(nmols)
Fig. 5. Effects of uric acid on the calibration curves of xanthine and hypoxanthine. Standard samples of xanthine and hypoxanthine. with or without adding uric acid (4 nmol). were carried through the entire standard assay procedure I“m the presence of uric acid” (see Method). Fluorescence was measured at a constant sensitivity throughout this experiment.
298
alkaline requiring subsequent neutralization. When directly connecting this preliminary treatment with the oxypurine-assay system, the hydrogen peroxide produced from the existing uric acid should be destroyed by the catalase involved in the oxypurine-assay system and the subsequent inactivation of the uricase should prevent the uric acid of the oxypurines-origin from being oxidized before the catalase treatment. Therefore, the preliminary treatment should not disturb the oxypurine assay if these processes properly function in the actual assay. This pretreating method was tested by examining the effects of added uric acid on the calibration curves of the oxypurines. The calibration curves were made according to the standard method “in the presence of uric acid”, with or without adding uric acid (4 nmol) to the standard samples of the oxypurines. The result (Fig. 5) shows that the uric acid added had no effect on the calibration curve of either xanthine or hypoxanthine. As we had preliminarily observed that the added uric acid disturbed the stoichiometry of the oxypurine assay when this uricase-treatment or the subsequent inactivation of this enzyme was omitted (data are not shown), the present result confirms that the proposed procedure actually removed contaminating uric acid without affecting the assay of oxypurines. Application to determine the total oxypurine content in the rat liver Since a fluorometric method is generally susceptible to the interference with contaminating fluorophors, the applicability of the present method was tested with the rat liver extract which is highly fluorescent and contains uric acid as well. The blank-fluorescence of perchloric acid-extract of the rat liver was really so strong that the direct assay on the extract was impossible. The interfering fluorophors, however, were not adsorbed onto the QAE-Sephadex A-25 column and almost completely washed away from the column with water. On the other hand, both xanthine and hypoxanthine were adsorbed and eluted from the column not with water but with 0.05 mol/l HCOOH. Through this column treatment, the fluorophors were completely removed; however, uric acid in the extract was not separated from the oxypurines. Before serving for the assay, the sample eluted from the column had to be evaporated to dryness for concentration and removing HCOOH. The assay was performed on the sample receiving this column treatment under the standard assay conditions “in the presence of uric acid”. The mean total content of oxypurines and the standard deviation obtained on 5 rats were determined to be 7.2 + 1.5 pmol/lOO g tissue. This content was lower than the reported chicken liver oxypurine level (23.8 + 8.9 pmol/lOO g tissue) [8] ; this being probably due to the species-specificity and (or) the different conditions of the tissue fixation because the oxypurines very rapidly increases immediately after death. The over all recovery with respect to the standard oxypurines added to the liver homogenate was 68-75s; the recoveries of xanthine and hypoxanthine were not different with each other. We have not, unfortunately, been able to test the present method on samples of human origin, however, this method with or without modification could be applied to human samples of blood, liver and muscle.
299
References 1 Guibault. G.G.. Brignac. P., Jr. and Zimmer, M. (1968) Anal. Chem. 40. 190-196 2 Klinkenberg, J.R., Goldfinger, S., Bradley, H.H. and Seegmiller, J.E. (1967) Clin. Chem. 3 Kalcker, H.M. (1947) J. Biol. Chem. 167, 429436 4 Bray. R.C. (1963) in The Enzymes (Bayer, pp. 533-556, Academic Press, New York 5 6 7 8
P.O..
Lardy.
H. and Myerback.
13. 834-846
K., eds.), 2nd edn.. Vol. 7,
Sumi, T., Umeda. Y., Kishi, Y., Takahashi. K. and Kakimoto, F. (1976) Clin. Chim. Acta Kuan, J.-C.W., Kuan. S.S. and Guilbault. G.G. (1975) Clin. Chim. Acta 64. 19-25 Truszkowski. R. (1930) Biochem. J. 23. 1359-1364 DeLapp, N.W. and Fisher. J.R. (1972) Biochim. Biophys. Acta 269, 505-514
73, 233-239
Clinica Chimica Acta, 95 (1979) 291-299 @ Elsevier/North-Holland Biomedical Press
CCA 1053
FLUOROMETRIC HYPOXANTHINE
TAKAHIKO
DETERMINATION IN TISSUE
OF XANTHINE
AND
SUM1 * and YURI UMEDA
Division of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-l-8, Kamikitazawa, Setagaya-Ku, Tokyo 156 (Japan) (Received
December
21st, 1978)
Summary A method was developed to determine the total content of the oxypurines, xanthine and hypoxanthine, in animal tissues. The developed method was constructed mainly from the following successive steps: (1) conversion of the oxypurines to uric acid and hydrogen peroxide by xanthine oxidase; (2) decomposition of the hydrogen peroxide by catalase and subsequent inactivation of this enzyme; (3) fluorometric measurement of the uric acid based on the coupled enzyme reaction of uricase and peroxidase. In applying this method to a sample containing uric acid, preliminary removal of this uric acid was necessary and this was carried out by treating the sample with uricase, followed by subsequent inactivation of this enzyme. The present method was more specific than the existing fluorometric method and permitted to measure the total content of the oxypurines (as low as 1 nmol) without mutual separation of them. The actual application of this method to the rat liver was demonstrated together with the method to prepare the tissue sample for the assay.
Introduction The measurement of the oxypurines, xanthine and hypoxanthine, in biological samples has been increasingly required for clinical or laboratory investigations on gout and other metabolic diseases related to puke metabolism. Spectrofluorometric [l] and spectrophotometric [ 2,3] methods by using xanthine oxidase have been developed for this purpose. Although the fluorometric method, which is principally a peroxidase-aided assay of the hydrogen peroxide produced from the oxypurines by xanthine oxidase, is simple and highly sensitive * To
whom
correspondence
should
be addressed.
292
[ 11, this method has two severe defects when applied to biological samples. The one is a lack of specificity because xanthine oxidase attacks not only xanthine and hypoxanthine but also other oxypurines, aldehydes, etc. [4], and the other one is a requirement of mutual separation of xanthine and hypoxanthine to determine a total content of their mixture because hydrogen peroxide produced is 1 mol/mol from xanthine and 2 mol/mol, from hypoxanthine. To overcome these defects, we have developed an improved fluorometric method which is constructed by connecting the conversion of the oxypurines to uric acid with the determination of the resultant uric acid by using uricase-peroxidase system [ 51. Principle The procedure of the developed method consists of the following successive steps: (1) conversion of the oxypurines to uric acid and hydrogen peroxide by xanthine oxidase, (2) destruction of the hydrogen peroxide by catalase, (3) heat-inactivation of the catalase, (4) conversion of the uric acid to allantoin and hydrogen peroxide by uricase and (5) fluorometry of the hydrogen peroxide from the uric acid by the peroxidase reaction coupled with a fluorophor, p-hydroxyphenylacetic acid; (4) with (5) has already been utilized by us to measure plasma uric acid [ 51. According to this procedure, hydrogen peroxide is produced successively from the oxypurines and the uric acid, whereas the former hydrogen peroxide. is destroyed by the catalase but the latter is no more destroyed by this enzyme because of the heat-inactivation. Therefore, the hydrogen peroxide to be estimated b,y the peroxidase system is not the one from the oxypurines themselves but the other one from their common metabolite, uric acid. As a result, one mol of both xanthine and hypoxanthine is determined finally as one mol of hydrogen peroxide, and the specificity of the production of the hydrogen peroxide to be estimated does not depend on the non-specific enzyme, xanthine oxidase, but on uricase which is highly specific for uric acid. These may promise to remove the potential defects of the existing fluorometric method. To apply this method to samples containing uric acid, a preliminary complete removal of the uric acid is necessary. In the proposed method, this is carried out by pretreating the samples with uricase, which is subsequently inactivated by heating to prevent this uricase from oxidizing the uric acid of the oxypurine-origin before the catalase treatment; the hydrogen peroxide produced by this pre-treatment is to be destroyed at the catalase step. Materials and methods Reagents Buffers. Phosphate buffer, 0.05 mol/l, pH 8.0; Tris-HCl buffer, 0.05 mol/l, pH 9.0. Xanthine oxidase solution. 100 munits of milk xanthine oxidase (0.75 units/ mg of protein) in 1.0 ml of the Tris-HCl buffer. This solution should be prepared every experiment. Catalase stock solution. 750 units of catalase (30 000 units/mg of solid) in
293
1.0 ml of 30% glycerol (with 10% ethanol). This is stable for at least 4 weeks at 4°C. Cat&se working solution. This solution (40 units/ml) is prepared by diluting the stock solution with the phosphate buffer. This should be prepared every experiment. Uricase solution. 15 munits of uricase (Type II, 15 units/g of solid) in 1.0 ml of the phosphate buffer. This is stable for at least 4 weeks in a frozen state (-20°C). Peroxidase stock solution. 200 units of horseradish peroxidase (195 units/mg of solid) in 1.0 ml of the phosphate buffer. This is preserved for at least 4 weeks in a frozen state (-20°C). p-hydroxyphenylucetic acid (pHPAA) stock solution. 4 mg of pHPAA in 1.0 ml of water. This solution is stable for at least 4 weeks in a frozen state (-20°C). pHPAA-peroxiduse-buffer solution. This is prepared by mixing 0.5 ml of the pHPAA stock solution, 0.5 ml of the peroxidase stock solution and 9.0 ml of the phosphate buffer. This is stable for at least 4 weeks in a frozen state (-20°C). Standard stock solutions. Xanthine solution, 1.0 mmol/l in the Trix-HCl 1.0 mmol/l in the Tris-HCl buffer; uric acid buffer; hypoxanthine solution, solution, 0.5 mmol/l in the phosphate buffer. These are stable for at least 4 weeks in a frozen state (-20°C). The working solutions of them are prepared by diluting the respective stock solutions with water. QAE-Sephudex A-25 formute form. QAE-Sephadex A-25 is regenerated and then converted to the formate ion. This is stored in the form of a slurry at 4°C. All the enzymes used here were purchased from Sigma Chemical Co., and QAE-Sephadex A-25 was from Pharmacia. Other chemicals were analytical grade commercial products. Appuru tus Fluorescence is measured with a Hitachi spectrofluorometer (MFP-4, Hitachi Ltd.) equipped with a xenon arc lamp. Slit widths for excitation and emission light were 5.0 and 10.0 nm, respectively. A Hitachi linear recorder was coupled with the fluorometer. Standard assay method in the absence of uric acid To 200 ~1 of oxypurine sample containing no uric acid, 80 ~1 of xanthine oxidase solution is added, and incubated for 15 min at 27°C. To the mixture, 20 ~1 of catalase solution is next added, and incubated for 20 min at 27°C. After the incubation, the mixture is heated for 10 min in a boiling-water bath to inactivate the catalase and then cooled in ice water. Next, 100 ~1 of uricase solution is added to the mixture and incubated for 30 min at 27°C. After adding 1.0 ml of pHPAA-peroxidase-buffer solution, the mixture is next incubated for 30 min at 27°C. After the incubation, the fluorescence is measured at an excitation wavelength of 317 nm and an emission wavelength of 414 nm [ 51. A sample in which xanthine oxidase solution is replaced by Tris-HCl buffer serves as a blank. The blank-fluorescence is subtracted from the sample-fluores-
294
cence, and this net increase intensity”.
will be represented
in the figures as “fluorescence
Standard assay method in the presence of uric acid To 200 ~1 of oxypurine sample containing uric acid, 100 .~l of uricase solution is added, and then incubated for 30 min at 27°C. This is next heated for 10 min in a boiling-water bath, and cooled in ice water. Then, the mixture is submitted to the oxypurine analysis; although the initial volume of the resulting oxypurine sample amounts to 300 ~1 after adding this &case treatment, the subsequent oxypurine assay is carried out according to the standard method without any modifications. Method for determining total oxypurine content in the rat liuer The rat liver extract is prepared and then submitted to the total oxypurine assay after removing interfering fluorescent substances from the extract. These preparative methods are described here. Immediately after decapitating the rat (male Wistar strain, 200-300 g body weight), the liver is removed and quickly frozen in liquid Nz. The frozen liver is homogenized with cold 0.6 mol/l HC104 (3 ml/g of tissue). The homogenate is centrifuged for 30 min at 6000 rpm, and the supernatant is served. The resultant precipitate is re-homogenized with cold 0.6 mol/l HC104 (1.5 ml/g tissue). The second homogenate is centrifuged as before, and the resultant supe~a~n~ is combined with the first. The pH of the combined extract is adjusted to 10.0 to 10.5, and the resultant precipitate is centrifuged off. l-ml aliquot from the resultarit supernatant is applied onto a QAE-Sephadex A-25 column (1.0 cm diameter, 5.0 cm long). Next, the column is washed with 12 ml of water and eluted with 12 ml of 0.05 mol/l HCOOH. The HCOOHeluent is evaporated to dryness in vacua. The resultant residue is dissolved in 1.0 ml of 0.005 mol/l NaOH. Two 0,2-ml aliquots from this solution are served each for the standard assay “in the presence of uric acid” and for the blank. Results and discussion Critical reactions in the assay procedure The critical steps in the proposed assay procedure may be the xanthine oxidase reaction, the catalase reaction and the inactivation of catalase. To check the actual progress of these reactions during the assay, the development of the fluorescence in a hypoxanthine sample (4 nmol) which was carried through the entire assay procedure was measured with varying only enzyme concentration or reaction time of the respective critical reactions. At first, the xanthine oxidase reaction was checked by varying the amount of xanthine oxidase; an exact period of the xanthine oxidase reaction is rather obscure because this enzyme is not inactivated even after the end of this reaction and may remain still active in the subsequent catalase reaction, and therefore the check was not done with respect to the t~~ou~e of xanthine oxidase reaction. As shown in Fig. 1, the fluorescence did not develop without this enzyme, increased markedly with increasing the amount of the enzyme and
295
o- 06
4 xanthine
8 12 oxidase (munits)
16
Fig. 1. Development 0:: fluorescence as a function of xanthine oxidase levels. Hypoxanthine samples (4 nmol) were carried through the entire standard assay procedure with only varying the amount of xanthine oxidase.
attained a plateau level throughout 2.0-16.0 munits of the enzyme. This indicates that the xanthine oxidase reaction actually occurred and the standard amount of xanthine oxidase was very sufficient to carry this reaction to the end under the standard assay conditions. The effect of the catalase treatment was next tested by following the change of the fluorescence as a function of the incubation time of catalase. The result (Fig. 2) shows that the fluorescence was in a maximum level without catalase treatment, then decreased with increasing the incubation time of catalase and attained a constant level throughout 15-25-min incubation. This suggests that the decreasing fluorescence was due to the hydrogen peroxide produced at the xanthine oxidase step, and the constant fluorescence reflected the other hydrogen peroxide produced at the uricase step. It should be noted that the catalase reaction went to completion within the standard 20-min incubation.
minutes Fig. 2. Development of fluorescence as a function (4 nmol) were carried through the entire standard with catalase.
of the time of catalase reaction. Hypoxanthine samples assay procedure with only varying the incubation time
296
0
2
4
6
8
10
12 minutes
Fig. 3. Development of fluorescence as a function of the time of heating for inactivating catalase. Hypoxanthine samples (4 nmol) were carried through the entire standard assay procedure with only varying the heating time for inactivating catalase.
Finally, the influence of the heat-inactivation of the catalase to the development of the fluorescence was examined by varying the heating time of the catalase in a boiling water bath. As shown in Fig. 3, the fluorescence did not develop without heating, but markedly increased by introducing this step of inactivation, the developed fluorescence being kept constant throughout 2-12 min heating. This result is consistent with a view that the catalase was capable of destroying both hydrogen peroxide produced from the oxypurine and from its metabolite, uric acid, but the latter hydrogen peroxide escaped the destruction by the preceeding heat-inactivation of this enzyme (compare with Fig. 2). In conclusion, it can be said that all of these critical reactions proceeded in the manner we had expected and almost completely ended under the standard assay conditions. Since the presented observations with these reactions were indirect, this conclusion is on the assumption that the uricase-peroxidase system was quantitatively coupled with the sequence of the critical reactions. This assumptions will be verified by the following results. Calibration curves Using the standard assay method described above, calibration curves were made with respect to the oxypurines. As shown in Fig. 4, the increase in fluorescence was proportional to concentrations of both xanthine and hypoxanthine, and these curves were completely identical with each other. This indicates that the proposed method can measure as low as 1 nmol of the oxypurines if the blank fluorescence is very low or is removed by a preliminary treatment of the sample, and that the measurement does not depend on the ratio between xanthine and hypoxanthine existing in the sample. Furthermore, the present result also gives an additional support to the view that the three critical reactions went to completion under the standard assay conditions, producing uric acid from the oxypurines and removing the hydrogen peroxide also produced from the oxypurines, thus the production of the uric acid was coupled quantitatively with the uricase-peroxidase system. It should be noted that the specificity of the present method is dependent
297
o hypoxanthine xanthine
l
oxypurines(nmols) Fig. 4. Calibration curves for xanthine and hypoxanthine. were carried through the entire standard assay procedure.
Standard
samples
of xanthine
and hypoxanthine
largely on the specificity of the uricase-peroxidase system, this system having been reported to be highly specific for uric acid [ $61 except for the interfere with ascorbic acid [6]. Preliminary
removal of contaminating
To remove uric acid adopted the preliminary sequent inactivation of inactivated by heating,
uric acid
existing very frequently in biological samples, we have treatment of the samples with uricase followed by subthis enzyme [ 71: in the present method, the uricase is which is simpler than the usual inactivation with strong
hypoxanthine(nmols)
xanthine(nmols)
Fig. 5. Effects of uric acid on the calibration curves of xanthine and hypoxanthine. Standard samples of xanthine and hypoxanthine. with or without adding uric acid (4 nmol). were carried through the entire standard assay procedure I“m the presence of uric acid” (see Method). Fluorescence was measured at a constant sensitivity throughout this experiment.
298
alkaline requiring subsequent neutralization. When directly connecting this preliminary treatment with the oxypurine-assay system, the hydrogen peroxide produced from the existing uric acid should be destroyed by the catalase involved in the oxypurine-assay system and the subsequent inactivation of the uricase should prevent the uric acid of the oxypurines-origin from being oxidized before the catalase treatment. Therefore, the preliminary treatment should not disturb the oxypurine assay if these processes properly function in the actual assay. This pretreating method was tested by examining the effects of added uric acid on the calibration curves of the oxypurines. The calibration curves were made according to the standard method “in the presence of uric acid”, with or without adding uric acid (4 nmol) to the standard samples of the oxypurines. The result (Fig. 5) shows that the uric acid added had no effect on the calibration curve of either xanthine or hypoxanthine. As we had preliminarily observed that the added uric acid disturbed the stoichiometry of the oxypurine assay when this uricase-treatment or the subsequent inactivation of this enzyme was omitted (data are not shown), the present result confirms that the proposed procedure actually removed contaminating uric acid without affecting the assay of oxypurines. Application to determine the total oxypurine content in the rat liver Since a fluorometric method is generally susceptible to the interference with contaminating fluorophors, the applicability of the present method was tested with the rat liver extract which is highly fluorescent and contains uric acid as well. The blank-fluorescence of perchloric acid-extract of the rat liver was really so strong that the direct assay on the extract was impossible. The interfering fluorophors, however, were not adsorbed onto the QAE-Sephadex A-25 column and almost completely washed away from the column with water. On the other hand, both xanthine and hypoxanthine were adsorbed and eluted from the column not with water but with 0.05 mol/l HCOOH. Through this column treatment, the fluorophors were completely removed; however, uric acid in the extract was not separated from the oxypurines. Before serving for the assay, the sample eluted from the column had to be evaporated to dryness for concentration and removing HCOOH. The assay was performed on the sample receiving this column treatment under the standard assay conditions “in the presence of uric acid”. The mean total content of oxypurines and the standard deviation obtained on 5 rats were determined to be 7.2 + 1.5 pmol/lOO g tissue. This content was lower than the reported chicken liver oxypurine level (23.8 + 8.9 pmol/lOO g tissue) [8] ; this being probably due to the species-specificity and (or) the different conditions of the tissue fixation because the oxypurines very rapidly increases immediately after death. The over all recovery with respect to the standard oxypurines added to the liver homogenate was 68-75s; the recoveries of xanthine and hypoxanthine were not different with each other. We have not, unfortunately, been able to test the present method on samples of human origin, however, this method with or without modification could be applied to human samples of blood, liver and muscle.
299
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