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Ascorbic acid suppresses the deconjugation of noradrenaline but not dopamine in plasma
ANALYTICAL
182,262-265
BIOCHEMISTRY
(1989)
Ascorbic Acid Suppresses the Deconjugation Noradrenaline but Not Dopamine in Plasma Mitsuko
Okada,
Kazunori
Mine,’
and Michihiro
of
Fujiwara*
Department of PsychosomaticMedicine, Faculty of Medicine, Kyushu University, Fukuoka 812, and *Department of Physiology and Pharmacology,Faculty of Pharmaceutical Sciences,Fukuoka University, Fukuoka 814-01,Japan
Received
December
13,19&X3
The characteristics of hydrolysis of sulfoconjugated noradrenaline (NA) and dopamine (DA) in plasma using sulfatase were investigated. Ascorbic acid has been used as an antioxidant during the hydrolysis of conjugated NA or DA. Hydrolysis of NA sulfates was considerably inhibited by adding ascorbic acid (0.5-10 mM), and slightly inhibited by adding dithiothreitol (l-10 mM). In contrast, the hydrolysis of DA sulfates was not affected after either ascorbic acid or DTT treatment. On the basis of these findings, the levels of NA sulfates previously reported are found to be markedly lower than the actual levels of NA sulfates in human plasma. 0 1989 Academic Press, Inc.
The measurement of free or sulfoconjugated noradrenaline (NA)’ and dopamine (DA) in plasma is evaluated as an index of central or sympathetic nervous activity in various clinical states (l-3). Most workers, in measuring sulfoconjugated NA and DA in plasma, frequently use ascorbic acid as a reducing agent to prevent catecholamine oxidation during hydrolysis procedures (45). However, it has been demonstrated that ascorbic acid reduces various enzyme activities in mammalian tissues (6-8). In the present study, we investigated the effects of an antioxidant, ascorbic acid, or dithiothreitol (DTT) on the hydrolysis of NA sulfates and DA sulfates in plasma with sulfatase. MATERIALS
AND
METHODS
Chemicals. Noradrenaline (NA) bitartrate, dopamine (DA) hydrochloride, 3,4-dihydroxybenzylamine (DHBA) hydrochloride, sulfatase type H-5, sulfatase type VI, L-ascorbic acid, DL-dithiothreitol (DTT) and 1 To whom correspondence should be addressed at the Department of Psychosomatic Medicine. ’ Abbreviations used: NA, noradrenaline; DA, dopamine; DTT, dithiothreitol; DHBA, 3,4-dihydroxybenzylamine. 262
pargyline hydrochloride were purchased from Sigma Chemical Company, St. Louis, Missouri, and alumina was from Bioanalytical Systems Inc., West Lafayette, Indiana. Other commercial chemicals and reagents were of analytical grade and were used without further purification. Double-distilled and deionized water (Barnstead NAN0 pure, Barnstead Company, Boston, Massachusetts) was used throughout. Sample preparation. Venous blood was collected from three to five healthy volunteers in heparinized polypropylene tubes. Plasma was promptly separated by centrifugation (5OOOg,20 min) and stored without preservatives at -80°C until analysis. After being thawed at room temperature, the plasma samples were stirred and 0.5 ml of each was transferred into an ice-cold polypropylene tube. Fifteen microliters of concentrated perchloric acid was then added into all tubes followed by 20 ~1of 10 mM EDTA- 2K medium (pH 7.2). The tubes were vortexed again and centrifugated at 10,OOOgfor 20 min. The supernatant was transferred into a new polypropylene tube. Excess perchlorate was precipitated by adding 200 ~1 of 2 M CH,COOK buffer (pH 6.5). The precipitate was separated by further centrifugation. Determination of NA and DA levels in plasma. Free NA and DA were measured after extraction with alumina by high-performance liquid chromatography (HPLC) with electrochemical detection according to Davies and Molyneux (9). Ten nanograms of DHBA was added to each sample just before the addition of perchloric acid. The data are expressed as means f SE. Recoveries of added 10 ng of NA, DHBA, and DA in 0.5 ml of plasma were 91.8 f 7.2, 84.4 f 7.1, and 79.6% f 8.3, respectively (n = 3). The levels of free NA and DA were 0.485 f 0.081 rig/ml and 0.092 _+0.001 rig/ml, respectively (n = 5). Total NA and DA were measured after enzymatic hydrolysis with 50 U of sulfatase type H-5 for 1-18 h, the 0003-2697/89
$3.00
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
AN’l‘IUXlJJAN’l
lNHlJ311
IUN
263
UP
final pH being about 5.0. Other procedures for measurement of total NA and DA were exactly the same as for the assay of free NA and DA. Levels of total NA and DA after incubation for 18 h in the absence of an antioxidant (control) were 7.50 + 0.23 and 12.99 + 5.76 rig/ml, respectively (n = 5). The average percentage free in control was 4.87 + 1.30% for NA and 0.74 f 0.21% for DA. The levels of hydrolyzed NA sulfate or DA sulfate in each sample were determined from the differences between the total and free levels. In some experiments, plasma samples were hydrolyzed for 18 h with 4 U of sulfatase type VI which is devoid of glucuronidase activity. Apparatus. The HPLC system used had four components: Waters 600 pump (Waters Associates, Inc.), U6K universal injector (Waters Associates, Inc.), EICOMPAK MA-ODS reversal phase column (EICOM CORP., Kyoto, Japan) with an in-line RCC Guard PAK packed Waters resolve Cl8 (Waters Associates, Inc.), and Waters 460 electrochemical detector (Waters Associates, Inc.). Chromatographic conditions. The mobile phase consisted of a mixture of 77 mM sodium monophosphate and 7.0% methanol containing 0.65 mM 1-octanesulfonic acid and 0.05 mM EDTA.2Na. The solution was adjusted to pH 4.05 by adding orthophosphoric acid and then was degassed under vacuum. Chromatography was carried out at room temperature at a mobile phase flow rate of 0.8 ml per minute while the flow cell potential was set at +0.70 V. RESULTS
Figure 1 shows the time course of the hydrolysis of plasma sulfoconjugated catecholamines in either the presence or the absence of 1 mM ascorbic acid. As shown in Fig. la, the shapes of the time courses of NA deconjugation under these conditions during the first 3 h were virtually identical. At 3 h after the beginning reaction, the progress curves began to diverge. The progress curve in the presence of 1 mM ascorbic acid apparently reached a steady-state level within 3 h. In contrast, for the incubation medium that contained no ascorbic acid, the time course for the hydrolysis of NA sulfates is suggestive of an initial phase extending for 3 h, followed by a phase in which NA deconjugation occurs at a slower rate. As shown in Fig. 2a, the levels of NA sulfates hydrolyzed with sulfatase for 3 h were approximately equal under all conditions. But in the presence of 1 mM ascorbic acid, the maximum level of NA sulfates after hydrolysis for 18 h was about a half of the level observed in the absence of ascorbic acid. The level of deconjugated NA after hydrolysis for 18 h in the absence of ascorbic acid was 7.09 +- 0.38 rig/ml (n = 5), and in the presence of 1 mM ascorbic acid, the level of deconjugated NA after 18 h hydrolysis was 3.03 + 0.23 rig/ml (n = 5). On the other hand, no significant alterations were observed during the hydrolysis of DA sulfates by 1 mM
a.
125
r 0
b.
125
I 3
6
9
12
15
16 hr.
3
6
9
12
15
16 hr.
r
25
0
Incubation
time
FIG. 1. Progress curves for the hydrolysis of NA sulfate (a) and DA sulfate (b) in plasma by sulfatase H-5 (50 U/ml) at pH 5 and 37°C: 0, in the presence of 1 mM ascorbic acid; 0, in the absence of ascorbic acid (control). Venous blood samples were collected from three healthy volunteers. The results represent the percentages of the control levels of deconjugated NA (a) and DA (b) after hydrolysis for 18 h. Data are expressed as means k SE. Student’s t test was used for statistical analysis. *P < 0.05, **P < 0.01: significant differences between the levels of deconjugated NA in the presence and absence of ascorbic acid.
ascorbic acid treatment (Fig. lb). The level of deconjugated DA after 18 h hydrolysis in the absence of ascorbic acid was 12.90 +-4.55 rig/ml (n = 5). The maximum levels of deconjugated DA in the presence of 1 mM ascorbic acid, 1 mM pargyline, or 1 mM DTT were almost identical to the control levels (Fig. 2b). Ascorbic acid (l-10 mM) inhibited the hydrolysis of NA sulfates by about 50-60%. Concentrations of ascorbic acid higher than 1 mM showed no greater inhibition of the hydrolysis of NA sulfates. DTT (l-10 mM) inhibited the hydrolysis of NA sulfates by about 25-30%. Concentrations of DTT higher than 1 mM promoted no greater suppression of the hydrolysis of NA sulfates. On the other hand, the hydrolysis of DA sulfates was not inhibited by the addition of ascorbic acid or DTT. It did, however, promote the hydrolysis of DA sulfates although no significant differences were seen (data not shown). Addition of a high concentration of antioxidant
264
OKADA, a.
150
AND
FUJIWARA 150
b.
3 hr. incubation 0
FIG.2.
MINE,
control
15 hr. Incubation 0
Effect of antioxidants and monoamine oxidase ascorbic acid, DTT, or pargyline or nothing was added to after 3 or 18 h of hydrolysis. Venous blood samples were Fig. 1. **P < 0.01: any significant differences are compared
1 mM pargyllnr
3 hr. incubation m
1 mM OTT
m
15 hr. Incubation I mM arcorblc
acid
on the hydrolysis of NA sulfates (a) and DA sulfates (b). A 1 mM a reaction mixture at the beginning of the hydrolysis. The reaction collected from five healthy volunteers. Conditions are as described to the level in the presence of 1 mM ascorbic acid.
caused no change in the pH values of any reaction mixtures. Also, we further examined the effects of ascorbic acid on the enzymatic hydrolysis of plasma conjugated NA using sulfatase type VI and arylsulfatase from Aerobatter aerogenes in which glucuronidase activity is not detected at pH 7.1. Although we hydrolyzed plasma NA sulfates at pH 5.0, the glucuronidase activity in plasma extracts with sulfatase type VI is much lower than that with sulfatase H-5. When the level of NA sulfates after 18 h hydrolysis in the absence of 1 mM ascorbic acid is set at lOO%, the level of NA sulfates after 18 h hydrolysis in the presence of ascorbic acid is 55.9 + 9.4% (n = 4). DISCUSSION
In most reports investigating plasma conjugated catecholamines, high concentrations of ascorbic acid were used as the antioxidant during deconjugation (3-5,lO). As shown in Fig. la, in the presence of a high concentration of ascorbic acid, the hydrolysis of NA sulfates is able to reach a steady-state level within 3 h. In addition, no significant differences are seen between the levels of plasma NA sulfates after 3 h hydrolysis in the absence and the presence of ascorbic acid. That may be one reason why the levels of plasma NA sulfates reported previously were measured after deconjugation for only less than 3 h (10-12). In the present study, the levels of deconjugated NA in plasma after hydrolysis for 18 h in the absence of an antioxidant (7.09 f 0.38 rig/ml, n = 5) were markedly higher than the levels previously reported by Wang et al. (0.40 f 0.04 rig/ml) (13) or Vlachakis et al.
concentration was terminated in the legend
of to
(0.559 f 0.097 rig/ml) (14). No significant difference was seen between the recovery of the standard mixture, NA, DHBA, and DA, which was added to plasma after 18 h of hydrolysis, and the recovery of standard mixture alone just before the incubation (data not shown). In addition, no significant differences were seen between the levels of plasma conjugated NA and DA after 18 h hydrolysis in the absence and the presence of a monoamine oxidase inhibitor, pargyline (Figs. 2a and b). In the absence of an antioxidant, monoamine oxidase contaminant in the reaction medium did not cause the oxidation of NA and DA. Therefore, to obtain the actual maximum levels of NA sulfates in plasma, a sample should be hydrolyzed for a longer period without antioxidant. The time course of the hydrolysis of DA sulfates under control conditions apparently shows a biphasic response (Fig. lb). This finding may suggest that more than two isomeric forms of DA sulfates exist in human plasma. In fact, DA sulfates in plasma are already known to exist in two isomeric forms, DA-30sulfate and DA-4-O-sulfate (15). According to Yoneda et al. (16) about 5.7% of total NA is NA glucuronate and the levels of sulfoconjugated NA are sixfold higher than the levels of NA glucuronate. In addition, we further examined the effect of ascorbic acid on the enzymatic hydrolysis of plasma conjugated NA using sulfatase type VI which is devoid of glucuronidase activity. When a purer sulfatase preparation was used, the hydrolysis of NA sulfates was inhibited by about 4050% in the presence of ascorbic acid. From these findings, the slow production of NA might be in part due to
ANTIOXIDANT
INHIBITION
OF
the formation of NA from NA glucuronide. However, the slow production of NA is due mostly to the formation of NA from NA sulfates, and no doubt, arylsulfatase is inhibited by ascorbic acid. Thus, partial inhibition of the enzymatic hydrolysis of NA sulfates by ascorbic acid may also indicate that one of the isomeric forms of NA sulfates cannot be hydrolyzed with sulfatase in the presence of ascorbic acid. By analogy to the previous findings on the isomeric forms of DA sulfates, two isomeric forms of NA sulfate, NA-3-O-sulfate and NA-4-O-sulfate, may also be present in human plasma. Ascorbic acid selectively inhibited the hydrolysis of NA sulfates, but did not affect the hydrolysis of DA sulfates (Fig. 1). Meek and Neff (17) have reported that in comparison with NA, DA has a much higher affinity for phenol sulfotransferase, which is responsible for the formation of sulfoconjuSimilarly, most DA sulfates gated catecholamines. might be more available substrates for arylsulfatase than NA sulfates. This may explain the selective inhibition of the enzymatic hydrolysis of NA sulfates by ascorbic acid. Although the mechanism of the inhibition of the hydrolysis of NA sulfates by the antioxidant is not clear, it is probable that the conformation of arylsulfatase may convert by addition of the antioxidant. Actually, it has been shown that at least three distinct sulfatases (arylsulfatases A, B, and C) with different substrate specificities and inhibitor sensitivities (l&19), exist in mammalian tissue, and that ascorbic acid is known to reduce the activities of arylsulfatases A and B (20). The existence in humans of a metabolic pathway that catalyzes the sulfate conjugation of catecholamines and their metabolites has been known for over 40 years (2123). The process of sulfoconjugation may play an important role in biogenic amine metabolism in the central nervous system of several species (24,25). Catecholamine sulfoconjugates may not only be inactivation products but may represent a storage or transport form of catecholamine, playing an important role in metabolic interconversion. Interestingly, Dunne et al. (26) reported that the increase in plasma conjugated NA after banana ingestion was inhibited by ascorbic acid treatment although the rise in conjugated DA was potentiated by ascorbic acid treatment. In the present study, ascorbic acid significantly inhibited the hydrolysis of NA sulfates in plasma and promoted the hydrolysis of DA sulfates, although significant differences were not seen. Therefore, it can be postulated that ascorbic acid might be involved in the regulation of the levels of catecholamines through conjugation and deconjugation, although much further work is required to better elucidate this speculation. In conclusion, ascorbic acid has been proven to reduce the hydrolysis of NA sulfates. Thus, the precise level of
NORADRENALINE
265
HYDROLYSIS
sulfoconjugated NA is much greater than that measured by previous methods in the presence of ascorbic acid used as an antioxidant. In addition, these findings indicate the possibility that ascorbic acid may modulate catecholamine metabolism. ACKNOWLEDGMENT We thank manuscript.
Dr.
B. Quinn
(Kyushu
University)
for proofreading
the
REFERENCES 1. Wyatt, R. J., Portnoy, B., Kupfer, D. J., Snyder, F., and Engelman, K. (1971) Arch. Gen. Psychiat. 24,65-70. 2. Kuchel, O., Buu, N. T., Fontaine, A., Hamet, P., Beroniade, V., Larochelle, P., and Genest, J. (1980) Hypertension 2,177-186. 3. Vandongen, R., Puddey, I. B., Beilin, L. J., Brand, son, L., and Rogers, P. (1985) Clin. Exp. Pharmacol.
G. R., DavidPhysiol. 12,
279-283. 4. Tyce, G. M., Sharpless, N. S., and Muenter, M. D. (1974) Clin. Pharmacol. Ther. 16,782-788. 5. Wang, P., Buu, N. T., Kuchel, O., and Genes& J. (1983) J. Lab. Clin. Med. 101,141-151. 6. Kanfer, J. N., and Spielvogel, 327,405-411.
C. H. (1973)
Biochim.
Biophys.
Acta
7. Hoehn, S. K., and Kanfer, J. N. (1978) Canud. J. Biochem. 66, 352-356. 8. Herman, H. H., Wimalasena, K., Fowler, L. C., Beard, C. A., and May, S. W. (1988) J. Biol. Chem. 263,666-672. 9. Davies, C. L., and Molyneux, S. G. (1982) J. Chromatgr. 231,4151. 10. Buu, N. T., and Kuchel, 0. (1977) J. Lab. Clin. Med. 90,680-685. 11. Peuler,
J. D., and Johnson,
12. Nouta, H., Mitsui, Sci. 2,303-308.
G. A. (1977)
A., Umegae,
Life Sci.
Y., and Ohkura,
21,625~636. Y. (1986)
13. Wang, P., Buu, N. T., Kuchel, O., and Genest, J. (1983) Clin. Med. 101,141-151. 14. Vlachakis, N. D., Kogosov, E., Yoneda, S., Alexander, Maronde, R. F. (1984) Clin. Chim. Acta, 199-209.
J. Lab. N., and
15. Elchisak, M. A. (1986) Fed. Proc. 46,2241-2246. 16. Yoneda, S., Alexander, N., and Vlachakis, N. D. (1983) 33,935-942. 17. Meek, J. L., and Neff, N. H. (1973) J. Neurochem. 21,1-9. 18. Whittemore, R. M., Pearce, L. B., and Roth, J. A. (1985) istry 24,2477-2402. 19. Weinshilboum, R. M. (1986) Fed. Proc. 45,2223-2228. 20. Leveille, C. R., and Schwartz, Res. 52,436-441.
E. R. (1982)
Life Sci.
Biochem-
Znt. J. Vitam.
21. Wengle, B. (1964) Actu Chem. Stand. l&65-76. 22. Foldes, A., and Meek, J. L. (1974) J. Neurochem.
Anal.
Nutr.
23,303-307.
23. Rivett, A. J., Eddy, B. J., and Roth, J. A. (1982) J. Neurochem. 39,1009-1016. 24. Roth, J. A., and Rivett, A. J. (1982) Biochem. Phurmacol. 31, 3017-3021. 25. Tyce, G. M., Messick, J. M., Yaksh, T. L., Byer, D. E., Danielson, D. R., and Rorie, D. K. (1986) Fed. Proc. 45,2247-2253. 26. Dunne, J. W., Davidson, L., Vandongen, R., Beilin, L. J., and Rogers, P. (1983) LifeSci. 33,1511-1517.
182,262-265
BIOCHEMISTRY
(1989)
Ascorbic Acid Suppresses the Deconjugation Noradrenaline but Not Dopamine in Plasma Mitsuko
Okada,
Kazunori
Mine,’
and Michihiro
of
Fujiwara*
Department of PsychosomaticMedicine, Faculty of Medicine, Kyushu University, Fukuoka 812, and *Department of Physiology and Pharmacology,Faculty of Pharmaceutical Sciences,Fukuoka University, Fukuoka 814-01,Japan
Received
December
13,19&X3
The characteristics of hydrolysis of sulfoconjugated noradrenaline (NA) and dopamine (DA) in plasma using sulfatase were investigated. Ascorbic acid has been used as an antioxidant during the hydrolysis of conjugated NA or DA. Hydrolysis of NA sulfates was considerably inhibited by adding ascorbic acid (0.5-10 mM), and slightly inhibited by adding dithiothreitol (l-10 mM). In contrast, the hydrolysis of DA sulfates was not affected after either ascorbic acid or DTT treatment. On the basis of these findings, the levels of NA sulfates previously reported are found to be markedly lower than the actual levels of NA sulfates in human plasma. 0 1989 Academic Press, Inc.
The measurement of free or sulfoconjugated noradrenaline (NA)’ and dopamine (DA) in plasma is evaluated as an index of central or sympathetic nervous activity in various clinical states (l-3). Most workers, in measuring sulfoconjugated NA and DA in plasma, frequently use ascorbic acid as a reducing agent to prevent catecholamine oxidation during hydrolysis procedures (45). However, it has been demonstrated that ascorbic acid reduces various enzyme activities in mammalian tissues (6-8). In the present study, we investigated the effects of an antioxidant, ascorbic acid, or dithiothreitol (DTT) on the hydrolysis of NA sulfates and DA sulfates in plasma with sulfatase. MATERIALS
AND
METHODS
Chemicals. Noradrenaline (NA) bitartrate, dopamine (DA) hydrochloride, 3,4-dihydroxybenzylamine (DHBA) hydrochloride, sulfatase type H-5, sulfatase type VI, L-ascorbic acid, DL-dithiothreitol (DTT) and 1 To whom correspondence should be addressed at the Department of Psychosomatic Medicine. ’ Abbreviations used: NA, noradrenaline; DA, dopamine; DTT, dithiothreitol; DHBA, 3,4-dihydroxybenzylamine. 262
pargyline hydrochloride were purchased from Sigma Chemical Company, St. Louis, Missouri, and alumina was from Bioanalytical Systems Inc., West Lafayette, Indiana. Other commercial chemicals and reagents were of analytical grade and were used without further purification. Double-distilled and deionized water (Barnstead NAN0 pure, Barnstead Company, Boston, Massachusetts) was used throughout. Sample preparation. Venous blood was collected from three to five healthy volunteers in heparinized polypropylene tubes. Plasma was promptly separated by centrifugation (5OOOg,20 min) and stored without preservatives at -80°C until analysis. After being thawed at room temperature, the plasma samples were stirred and 0.5 ml of each was transferred into an ice-cold polypropylene tube. Fifteen microliters of concentrated perchloric acid was then added into all tubes followed by 20 ~1of 10 mM EDTA- 2K medium (pH 7.2). The tubes were vortexed again and centrifugated at 10,OOOgfor 20 min. The supernatant was transferred into a new polypropylene tube. Excess perchlorate was precipitated by adding 200 ~1 of 2 M CH,COOK buffer (pH 6.5). The precipitate was separated by further centrifugation. Determination of NA and DA levels in plasma. Free NA and DA were measured after extraction with alumina by high-performance liquid chromatography (HPLC) with electrochemical detection according to Davies and Molyneux (9). Ten nanograms of DHBA was added to each sample just before the addition of perchloric acid. The data are expressed as means f SE. Recoveries of added 10 ng of NA, DHBA, and DA in 0.5 ml of plasma were 91.8 f 7.2, 84.4 f 7.1, and 79.6% f 8.3, respectively (n = 3). The levels of free NA and DA were 0.485 f 0.081 rig/ml and 0.092 _+0.001 rig/ml, respectively (n = 5). Total NA and DA were measured after enzymatic hydrolysis with 50 U of sulfatase type H-5 for 1-18 h, the 0003-2697/89
$3.00
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
AN’l‘IUXlJJAN’l
lNHlJ311
IUN
263
UP
final pH being about 5.0. Other procedures for measurement of total NA and DA were exactly the same as for the assay of free NA and DA. Levels of total NA and DA after incubation for 18 h in the absence of an antioxidant (control) were 7.50 + 0.23 and 12.99 + 5.76 rig/ml, respectively (n = 5). The average percentage free in control was 4.87 + 1.30% for NA and 0.74 f 0.21% for DA. The levels of hydrolyzed NA sulfate or DA sulfate in each sample were determined from the differences between the total and free levels. In some experiments, plasma samples were hydrolyzed for 18 h with 4 U of sulfatase type VI which is devoid of glucuronidase activity. Apparatus. The HPLC system used had four components: Waters 600 pump (Waters Associates, Inc.), U6K universal injector (Waters Associates, Inc.), EICOMPAK MA-ODS reversal phase column (EICOM CORP., Kyoto, Japan) with an in-line RCC Guard PAK packed Waters resolve Cl8 (Waters Associates, Inc.), and Waters 460 electrochemical detector (Waters Associates, Inc.). Chromatographic conditions. The mobile phase consisted of a mixture of 77 mM sodium monophosphate and 7.0% methanol containing 0.65 mM 1-octanesulfonic acid and 0.05 mM EDTA.2Na. The solution was adjusted to pH 4.05 by adding orthophosphoric acid and then was degassed under vacuum. Chromatography was carried out at room temperature at a mobile phase flow rate of 0.8 ml per minute while the flow cell potential was set at +0.70 V. RESULTS
Figure 1 shows the time course of the hydrolysis of plasma sulfoconjugated catecholamines in either the presence or the absence of 1 mM ascorbic acid. As shown in Fig. la, the shapes of the time courses of NA deconjugation under these conditions during the first 3 h were virtually identical. At 3 h after the beginning reaction, the progress curves began to diverge. The progress curve in the presence of 1 mM ascorbic acid apparently reached a steady-state level within 3 h. In contrast, for the incubation medium that contained no ascorbic acid, the time course for the hydrolysis of NA sulfates is suggestive of an initial phase extending for 3 h, followed by a phase in which NA deconjugation occurs at a slower rate. As shown in Fig. 2a, the levels of NA sulfates hydrolyzed with sulfatase for 3 h were approximately equal under all conditions. But in the presence of 1 mM ascorbic acid, the maximum level of NA sulfates after hydrolysis for 18 h was about a half of the level observed in the absence of ascorbic acid. The level of deconjugated NA after hydrolysis for 18 h in the absence of ascorbic acid was 7.09 +- 0.38 rig/ml (n = 5), and in the presence of 1 mM ascorbic acid, the level of deconjugated NA after 18 h hydrolysis was 3.03 + 0.23 rig/ml (n = 5). On the other hand, no significant alterations were observed during the hydrolysis of DA sulfates by 1 mM
a.
125
r 0
b.
125
I 3
6
9
12
15
16 hr.
3
6
9
12
15
16 hr.
r
25
0
Incubation
time
FIG. 1. Progress curves for the hydrolysis of NA sulfate (a) and DA sulfate (b) in plasma by sulfatase H-5 (50 U/ml) at pH 5 and 37°C: 0, in the presence of 1 mM ascorbic acid; 0, in the absence of ascorbic acid (control). Venous blood samples were collected from three healthy volunteers. The results represent the percentages of the control levels of deconjugated NA (a) and DA (b) after hydrolysis for 18 h. Data are expressed as means k SE. Student’s t test was used for statistical analysis. *P < 0.05, **P < 0.01: significant differences between the levels of deconjugated NA in the presence and absence of ascorbic acid.
ascorbic acid treatment (Fig. lb). The level of deconjugated DA after 18 h hydrolysis in the absence of ascorbic acid was 12.90 +-4.55 rig/ml (n = 5). The maximum levels of deconjugated DA in the presence of 1 mM ascorbic acid, 1 mM pargyline, or 1 mM DTT were almost identical to the control levels (Fig. 2b). Ascorbic acid (l-10 mM) inhibited the hydrolysis of NA sulfates by about 50-60%. Concentrations of ascorbic acid higher than 1 mM showed no greater inhibition of the hydrolysis of NA sulfates. DTT (l-10 mM) inhibited the hydrolysis of NA sulfates by about 25-30%. Concentrations of DTT higher than 1 mM promoted no greater suppression of the hydrolysis of NA sulfates. On the other hand, the hydrolysis of DA sulfates was not inhibited by the addition of ascorbic acid or DTT. It did, however, promote the hydrolysis of DA sulfates although no significant differences were seen (data not shown). Addition of a high concentration of antioxidant
264
OKADA, a.
150
AND
FUJIWARA 150
b.
3 hr. incubation 0
FIG.2.
MINE,
control
15 hr. Incubation 0
Effect of antioxidants and monoamine oxidase ascorbic acid, DTT, or pargyline or nothing was added to after 3 or 18 h of hydrolysis. Venous blood samples were Fig. 1. **P < 0.01: any significant differences are compared
1 mM pargyllnr
3 hr. incubation m
1 mM OTT
m
15 hr. Incubation I mM arcorblc
acid
on the hydrolysis of NA sulfates (a) and DA sulfates (b). A 1 mM a reaction mixture at the beginning of the hydrolysis. The reaction collected from five healthy volunteers. Conditions are as described to the level in the presence of 1 mM ascorbic acid.
caused no change in the pH values of any reaction mixtures. Also, we further examined the effects of ascorbic acid on the enzymatic hydrolysis of plasma conjugated NA using sulfatase type VI and arylsulfatase from Aerobatter aerogenes in which glucuronidase activity is not detected at pH 7.1. Although we hydrolyzed plasma NA sulfates at pH 5.0, the glucuronidase activity in plasma extracts with sulfatase type VI is much lower than that with sulfatase H-5. When the level of NA sulfates after 18 h hydrolysis in the absence of 1 mM ascorbic acid is set at lOO%, the level of NA sulfates after 18 h hydrolysis in the presence of ascorbic acid is 55.9 + 9.4% (n = 4). DISCUSSION
In most reports investigating plasma conjugated catecholamines, high concentrations of ascorbic acid were used as the antioxidant during deconjugation (3-5,lO). As shown in Fig. la, in the presence of a high concentration of ascorbic acid, the hydrolysis of NA sulfates is able to reach a steady-state level within 3 h. In addition, no significant differences are seen between the levels of plasma NA sulfates after 3 h hydrolysis in the absence and the presence of ascorbic acid. That may be one reason why the levels of plasma NA sulfates reported previously were measured after deconjugation for only less than 3 h (10-12). In the present study, the levels of deconjugated NA in plasma after hydrolysis for 18 h in the absence of an antioxidant (7.09 f 0.38 rig/ml, n = 5) were markedly higher than the levels previously reported by Wang et al. (0.40 f 0.04 rig/ml) (13) or Vlachakis et al.
concentration was terminated in the legend
of to
(0.559 f 0.097 rig/ml) (14). No significant difference was seen between the recovery of the standard mixture, NA, DHBA, and DA, which was added to plasma after 18 h of hydrolysis, and the recovery of standard mixture alone just before the incubation (data not shown). In addition, no significant differences were seen between the levels of plasma conjugated NA and DA after 18 h hydrolysis in the absence and the presence of a monoamine oxidase inhibitor, pargyline (Figs. 2a and b). In the absence of an antioxidant, monoamine oxidase contaminant in the reaction medium did not cause the oxidation of NA and DA. Therefore, to obtain the actual maximum levels of NA sulfates in plasma, a sample should be hydrolyzed for a longer period without antioxidant. The time course of the hydrolysis of DA sulfates under control conditions apparently shows a biphasic response (Fig. lb). This finding may suggest that more than two isomeric forms of DA sulfates exist in human plasma. In fact, DA sulfates in plasma are already known to exist in two isomeric forms, DA-30sulfate and DA-4-O-sulfate (15). According to Yoneda et al. (16) about 5.7% of total NA is NA glucuronate and the levels of sulfoconjugated NA are sixfold higher than the levels of NA glucuronate. In addition, we further examined the effect of ascorbic acid on the enzymatic hydrolysis of plasma conjugated NA using sulfatase type VI which is devoid of glucuronidase activity. When a purer sulfatase preparation was used, the hydrolysis of NA sulfates was inhibited by about 4050% in the presence of ascorbic acid. From these findings, the slow production of NA might be in part due to
ANTIOXIDANT
INHIBITION
OF
the formation of NA from NA glucuronide. However, the slow production of NA is due mostly to the formation of NA from NA sulfates, and no doubt, arylsulfatase is inhibited by ascorbic acid. Thus, partial inhibition of the enzymatic hydrolysis of NA sulfates by ascorbic acid may also indicate that one of the isomeric forms of NA sulfates cannot be hydrolyzed with sulfatase in the presence of ascorbic acid. By analogy to the previous findings on the isomeric forms of DA sulfates, two isomeric forms of NA sulfate, NA-3-O-sulfate and NA-4-O-sulfate, may also be present in human plasma. Ascorbic acid selectively inhibited the hydrolysis of NA sulfates, but did not affect the hydrolysis of DA sulfates (Fig. 1). Meek and Neff (17) have reported that in comparison with NA, DA has a much higher affinity for phenol sulfotransferase, which is responsible for the formation of sulfoconjuSimilarly, most DA sulfates gated catecholamines. might be more available substrates for arylsulfatase than NA sulfates. This may explain the selective inhibition of the enzymatic hydrolysis of NA sulfates by ascorbic acid. Although the mechanism of the inhibition of the hydrolysis of NA sulfates by the antioxidant is not clear, it is probable that the conformation of arylsulfatase may convert by addition of the antioxidant. Actually, it has been shown that at least three distinct sulfatases (arylsulfatases A, B, and C) with different substrate specificities and inhibitor sensitivities (l&19), exist in mammalian tissue, and that ascorbic acid is known to reduce the activities of arylsulfatases A and B (20). The existence in humans of a metabolic pathway that catalyzes the sulfate conjugation of catecholamines and their metabolites has been known for over 40 years (2123). The process of sulfoconjugation may play an important role in biogenic amine metabolism in the central nervous system of several species (24,25). Catecholamine sulfoconjugates may not only be inactivation products but may represent a storage or transport form of catecholamine, playing an important role in metabolic interconversion. Interestingly, Dunne et al. (26) reported that the increase in plasma conjugated NA after banana ingestion was inhibited by ascorbic acid treatment although the rise in conjugated DA was potentiated by ascorbic acid treatment. In the present study, ascorbic acid significantly inhibited the hydrolysis of NA sulfates in plasma and promoted the hydrolysis of DA sulfates, although significant differences were not seen. Therefore, it can be postulated that ascorbic acid might be involved in the regulation of the levels of catecholamines through conjugation and deconjugation, although much further work is required to better elucidate this speculation. In conclusion, ascorbic acid has been proven to reduce the hydrolysis of NA sulfates. Thus, the precise level of
NORADRENALINE
265
HYDROLYSIS
sulfoconjugated NA is much greater than that measured by previous methods in the presence of ascorbic acid used as an antioxidant. In addition, these findings indicate the possibility that ascorbic acid may modulate catecholamine metabolism. ACKNOWLEDGMENT We thank manuscript.
Dr.
B. Quinn
(Kyushu
University)
for proofreading
the
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