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Spectrofluorimetric determination of ascorbic acid
Analytica Chimica Acta, 161 (1983)
251-254 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
SPECTROFLUORIMETRIC
CHENOLI P. SHEELA,*
Department (India)
DETERMINATION
OF ASCORBIC ACID
ELIMBAN VIJAYANb and ABBURI RAMAIAH*
of Biochemistry,
All India Institute of Medical Sciences, New Delhi-l 10029
(Received 30th July 1982)
Summary. The spectrofluorimetric method described for the determination of > 20 nmol of ascorbic acid is based on its ability to participate in the nonenzymatic hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (dopa). The method is applied to Dulbeco’s culture media.
The principal methods for the determination of ascorbic acid involve titration with a reducible dye such as 2,6dichlorophenolindophenol or the formation of an osazone with 2,4dinitrophenylhydrazine [l]. During the determination of tyrosinase in human skin [2] by spectrofluorimetric measurement of 3,4dihydroxyphenylalanine (dopa) formed in the presence of the enzyme, tyrosine and ascorbic acid, it was observed that dopa was also formed nonenzymatically in significant quantities under certain conditions. The concentration of dopa thus formed was proportional to the concentrations of tyrosine and ascorbic acid. This reaction is perhaps similar to the hydroxylation of aromatic compounds in the presence of oxygen and iron(I1) [3] . This communication describes a method for the determination of 220 nmol of ascorbic acid based on this reaction, and shows that EDTA and iron(I1) are required to maximize the hydroxylation of tyrosine to dopa. Experimental L-Tyrosine, Ldopa and L-ascorbic acid were obtained from the Sigma Chemical Co. All other chemicals used were of analytical-reagent grade. LAscorbic acid solution was always freshly made up in 0.06% metaphosphoric acid. L-Tyrosine was made up in 0.2 M phosphate buffer, pH 6.8. A Farrand manual spectrofluorimeter was used for all fluorescence measurements. Procedure. The reaction was carried out in rimless tubes (12 mm diameter, 100 mm tall). The reaction mixture was 0.5 ml of 5 mM tyrosine in 0.2 M phosphate buffer, pH 6.8, 0.05 ml of 1.3 mM iron(I1) sulphate, 0.05 ml of 6.5 mM EDTA, 0.1 ml of 2 mM ascorbic acid, 0.1 ml of 0.6% metaphosPresent addresses: aPrabhalayam, Kakkad, Cannanore-5, Kerala (India); bDepartment Physiology, University of Manitoba, Faculty of Medicine, Winnipeg (Canada). 0003-2670/83/$03.00
o 1983 Elsevier Science Publishers B.V.
of
252
phoric acid and 0.2 ml of water to make the total volume 1 ml. The pH of this solution was 6.5. The mixture was incubated at 37°C in a water bath for 30 min. At the end of the reaction, 50 ~1 of the reaction mixture was added to 0.90 ml of 10 mM phosphate buffer pH 6.5 containing 0.0025% zinc sulphate followed by 0.1 ml of 0.25% potassium hexacyanoferrate(II1). The oxidation of dopa by hexacyanoferrate(II1) was terminated after exactly 2 min by addition of 0.1 ml of a freshly made mixture of 5 M sodium hydroxide and 2% ascorbic acid (9:l v/v). After 5 min, the fluorescence of the sample was measured at 360 nm excitation and 490 nm emission wavelengths. The chemistry involved in the estimation of dopa by this method has been described [4]. During the experiments, the sensitivity of the instrument fluctuated slightly. The instrument was standardized periodically with a 50 ng ml-’ solution of quinine sulphate in 0.05 M sulphuric acid. The fluorescence obtained by a number of standard solutions of dopa (30-20,000 pmol) gave a linear calibration graph, which was used to determine the ascorbic acid originally present. Such a graph was obtained periodically, but for daily use, in every experiment the fluorescence obtained from 50 ~1 of freshly made 11 X 10” M dopa was used to calculate the amount of dopa resulting from the presence of ascorbic acid. Results and discussion The product formed nonenzymatically in the presence of tyrosine and ascorbic acid was shown to be 3,4dihydroxyphenylaIanine (dopa) [2] . This was determined as described originally by Bertler et al. [4] as modified by Adachi and Halprin [ 51 and Husain et al. [2]. The components required for the maximal rate of nonenzymatic hydroxylation of tyrosine were studied. The fluorescence arising from dopa was obtained in the presence or absence of ascorbic acid, EDTA or iron(I1) sulphate or in the absence of both iron(I1) sulphate and EDTA. The fluorescence from dopa in the absence of ascorbic acid is very small (2% of the fluorescence obtained in presence of 0.2 mM ascorbic acid), showing thereby that the formation of dopa is almost totally dependent on the presence of ascorbic acid. In the absence of EDTA or iron sulphate, the fluorescence is decreased to 25% of its value in their presence. In the absence of both, the fluorescence was decreased by a further 15%. These results suggest that both EDTA and iron sulphate are required to enhance the rate of hydroxylation of tyrosine by ascorbic acid. The addition of EDTA and iron(I1) sulphate above the concentrations mentioned in the procedure did not increase the fluorescence. These experiments were done always in duplicate and the variation was less than 10%. Effect of ascorbic acid concentration. The rates of’formation of dopa at two different concentrations of ascorbic acid are shown in Fig. 1. The change in fluorescence is proportional to the ascorbic acid concentration for at least 30 min. The amount of dopa formed is directly proportional to the amount of ascorbic acid (100 nmol ascorbic acid = ca. 25 nmol dopa) up to
253
15
30
15
60
REACTION TIME (MINI
Fig. 1. Rate of formation of dopa in the presence of the following concentrations ascorbic acid: (0) 0.02 r&I; (0) 0.10 M (conditions otherwise as in procedure).
of
ca. 200 nmol of ascorbic acid. When the reaction was conducted in wider tubes (25 mm diameter), the dopa formed was proportional up to 400 nmol of ascorbic acid. It appears that at higher concentrations of ascorbic acid the reaction may be limited by the availability of oxygen. Precision and recovery. The relative standard deviation for a determination of 100 run01 of ascorbic acid was 3% (n = 8). Ascorbic acid was added to Dulbeco’s Modified Eagle’s Medium [6] to give a concentration of 2 mM, and five different aliquots of this medium were added to the reaction mixture described earlier to give a range of concentration of ascorbic acid from 0.02 to 0.2 mM in the reaction mixture. The recovery of duplicate determinations in each case was 98 + 3%. The method can be applied to the determination of ascorbic acid in the range 0.02-0.2 mM in the reaction mixture. Since only 50 ~1 of reaction mixture was used for the determination of dopa, the reaction can equally well be conducted in a 50-~1 volume and all of it can be used for the determination of dopa, in which case the method can detect as little as 1 nmol of ascorbic acid. Mechanism of hydroxylution of tyrosine. Ascorbic acid, by its auto-oxidation in the presence of oxygen, results in the formation of hydrogen peroxide which is reduced by iron(I1) complexes to form hydroxyl radicals [ 71. The hydroxyl radicals hydroxylate aromatic compounds [ 8 ] . This hydroxylation is stimulated by ascorbic acid by an as yet unknown mechanism. This work was supported by grants from the Department of Science and Technology, India (HCS/DST/971/80) to A. Ramaiah. The authors thank Miss Chaya Devi and Miss Aarti Bhatnagar for doing the reproducibility experiments.
254 REFERENCES 1 J. H. Roe, in P. Gyorgy and W. N. Pearson (Eds.), The Vitamins, 2nd edn., Vol. VII, Academic Press, New York, 1967, p. 27. 2 I. Husain, E. Vijayan, A. Ramaiah, J. S. Pasricha and N. C. Madan, J. Invest. Dermatol., 78 (1982) 243. 3 S. Udenfriend, C. J. Clark, J. Axeirod and B. B. Brodie, J. Biol. Chem., 208 (1954) 731. 4 A. Bertier, A. Carlsson and E. Rosengren, Acta Physiol. Stand., 44 (1958) 273. 5 K. Adachi and K. M. Halprin, Biochem. Biophys. Res. Commun., 26 (1967) 241. 6 R. Dulbeco and G. Freeman, Virology, 8 (1959) 396. 7 W. T. Dixon and R. 0. C. Norman, Nature, 196 (1962) 891. 8 W. T. Dixon and R. 0. C. Norman, Proc. Chem. Sot. (London), (1963) 97.
251-254 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
SPECTROFLUORIMETRIC
CHENOLI P. SHEELA,*
Department (India)
DETERMINATION
OF ASCORBIC ACID
ELIMBAN VIJAYANb and ABBURI RAMAIAH*
of Biochemistry,
All India Institute of Medical Sciences, New Delhi-l 10029
(Received 30th July 1982)
Summary. The spectrofluorimetric method described for the determination of > 20 nmol of ascorbic acid is based on its ability to participate in the nonenzymatic hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (dopa). The method is applied to Dulbeco’s culture media.
The principal methods for the determination of ascorbic acid involve titration with a reducible dye such as 2,6dichlorophenolindophenol or the formation of an osazone with 2,4dinitrophenylhydrazine [l]. During the determination of tyrosinase in human skin [2] by spectrofluorimetric measurement of 3,4dihydroxyphenylalanine (dopa) formed in the presence of the enzyme, tyrosine and ascorbic acid, it was observed that dopa was also formed nonenzymatically in significant quantities under certain conditions. The concentration of dopa thus formed was proportional to the concentrations of tyrosine and ascorbic acid. This reaction is perhaps similar to the hydroxylation of aromatic compounds in the presence of oxygen and iron(I1) [3] . This communication describes a method for the determination of 220 nmol of ascorbic acid based on this reaction, and shows that EDTA and iron(I1) are required to maximize the hydroxylation of tyrosine to dopa. Experimental L-Tyrosine, Ldopa and L-ascorbic acid were obtained from the Sigma Chemical Co. All other chemicals used were of analytical-reagent grade. LAscorbic acid solution was always freshly made up in 0.06% metaphosphoric acid. L-Tyrosine was made up in 0.2 M phosphate buffer, pH 6.8. A Farrand manual spectrofluorimeter was used for all fluorescence measurements. Procedure. The reaction was carried out in rimless tubes (12 mm diameter, 100 mm tall). The reaction mixture was 0.5 ml of 5 mM tyrosine in 0.2 M phosphate buffer, pH 6.8, 0.05 ml of 1.3 mM iron(I1) sulphate, 0.05 ml of 6.5 mM EDTA, 0.1 ml of 2 mM ascorbic acid, 0.1 ml of 0.6% metaphosPresent addresses: aPrabhalayam, Kakkad, Cannanore-5, Kerala (India); bDepartment Physiology, University of Manitoba, Faculty of Medicine, Winnipeg (Canada). 0003-2670/83/$03.00
o 1983 Elsevier Science Publishers B.V.
of
252
phoric acid and 0.2 ml of water to make the total volume 1 ml. The pH of this solution was 6.5. The mixture was incubated at 37°C in a water bath for 30 min. At the end of the reaction, 50 ~1 of the reaction mixture was added to 0.90 ml of 10 mM phosphate buffer pH 6.5 containing 0.0025% zinc sulphate followed by 0.1 ml of 0.25% potassium hexacyanoferrate(II1). The oxidation of dopa by hexacyanoferrate(II1) was terminated after exactly 2 min by addition of 0.1 ml of a freshly made mixture of 5 M sodium hydroxide and 2% ascorbic acid (9:l v/v). After 5 min, the fluorescence of the sample was measured at 360 nm excitation and 490 nm emission wavelengths. The chemistry involved in the estimation of dopa by this method has been described [4]. During the experiments, the sensitivity of the instrument fluctuated slightly. The instrument was standardized periodically with a 50 ng ml-’ solution of quinine sulphate in 0.05 M sulphuric acid. The fluorescence obtained by a number of standard solutions of dopa (30-20,000 pmol) gave a linear calibration graph, which was used to determine the ascorbic acid originally present. Such a graph was obtained periodically, but for daily use, in every experiment the fluorescence obtained from 50 ~1 of freshly made 11 X 10” M dopa was used to calculate the amount of dopa resulting from the presence of ascorbic acid. Results and discussion The product formed nonenzymatically in the presence of tyrosine and ascorbic acid was shown to be 3,4dihydroxyphenylaIanine (dopa) [2] . This was determined as described originally by Bertler et al. [4] as modified by Adachi and Halprin [ 51 and Husain et al. [2]. The components required for the maximal rate of nonenzymatic hydroxylation of tyrosine were studied. The fluorescence arising from dopa was obtained in the presence or absence of ascorbic acid, EDTA or iron(I1) sulphate or in the absence of both iron(I1) sulphate and EDTA. The fluorescence from dopa in the absence of ascorbic acid is very small (2% of the fluorescence obtained in presence of 0.2 mM ascorbic acid), showing thereby that the formation of dopa is almost totally dependent on the presence of ascorbic acid. In the absence of EDTA or iron sulphate, the fluorescence is decreased to 25% of its value in their presence. In the absence of both, the fluorescence was decreased by a further 15%. These results suggest that both EDTA and iron sulphate are required to enhance the rate of hydroxylation of tyrosine by ascorbic acid. The addition of EDTA and iron(I1) sulphate above the concentrations mentioned in the procedure did not increase the fluorescence. These experiments were done always in duplicate and the variation was less than 10%. Effect of ascorbic acid concentration. The rates of’formation of dopa at two different concentrations of ascorbic acid are shown in Fig. 1. The change in fluorescence is proportional to the ascorbic acid concentration for at least 30 min. The amount of dopa formed is directly proportional to the amount of ascorbic acid (100 nmol ascorbic acid = ca. 25 nmol dopa) up to
253
15
30
15
60
REACTION TIME (MINI
Fig. 1. Rate of formation of dopa in the presence of the following concentrations ascorbic acid: (0) 0.02 r&I; (0) 0.10 M (conditions otherwise as in procedure).
of
ca. 200 nmol of ascorbic acid. When the reaction was conducted in wider tubes (25 mm diameter), the dopa formed was proportional up to 400 nmol of ascorbic acid. It appears that at higher concentrations of ascorbic acid the reaction may be limited by the availability of oxygen. Precision and recovery. The relative standard deviation for a determination of 100 run01 of ascorbic acid was 3% (n = 8). Ascorbic acid was added to Dulbeco’s Modified Eagle’s Medium [6] to give a concentration of 2 mM, and five different aliquots of this medium were added to the reaction mixture described earlier to give a range of concentration of ascorbic acid from 0.02 to 0.2 mM in the reaction mixture. The recovery of duplicate determinations in each case was 98 + 3%. The method can be applied to the determination of ascorbic acid in the range 0.02-0.2 mM in the reaction mixture. Since only 50 ~1 of reaction mixture was used for the determination of dopa, the reaction can equally well be conducted in a 50-~1 volume and all of it can be used for the determination of dopa, in which case the method can detect as little as 1 nmol of ascorbic acid. Mechanism of hydroxylution of tyrosine. Ascorbic acid, by its auto-oxidation in the presence of oxygen, results in the formation of hydrogen peroxide which is reduced by iron(I1) complexes to form hydroxyl radicals [ 71. The hydroxyl radicals hydroxylate aromatic compounds [ 8 ] . This hydroxylation is stimulated by ascorbic acid by an as yet unknown mechanism. This work was supported by grants from the Department of Science and Technology, India (HCS/DST/971/80) to A. Ramaiah. The authors thank Miss Chaya Devi and Miss Aarti Bhatnagar for doing the reproducibility experiments.
254 REFERENCES 1 J. H. Roe, in P. Gyorgy and W. N. Pearson (Eds.), The Vitamins, 2nd edn., Vol. VII, Academic Press, New York, 1967, p. 27. 2 I. Husain, E. Vijayan, A. Ramaiah, J. S. Pasricha and N. C. Madan, J. Invest. Dermatol., 78 (1982) 243. 3 S. Udenfriend, C. J. Clark, J. Axeirod and B. B. Brodie, J. Biol. Chem., 208 (1954) 731. 4 A. Bertier, A. Carlsson and E. Rosengren, Acta Physiol. Stand., 44 (1958) 273. 5 K. Adachi and K. M. Halprin, Biochem. Biophys. Res. Commun., 26 (1967) 241. 6 R. Dulbeco and G. Freeman, Virology, 8 (1959) 396. 7 W. T. Dixon and R. 0. C. Norman, Nature, 196 (1962) 891. 8 W. T. Dixon and R. 0. C. Norman, Proc. Chem. Sot. (London), (1963) 97.