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Free Radical Biology & Medicine, Vol. 18, No. 5, pp. 955-956, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved

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Letters to the Editors

COMMENTS ON THE GLUTATHIONE-ASCORBIC ACID REDOX COUPLE

To the Editors:

We read with interest the review article on "The redox couple between glutathione and ascorbic acid: A chem- ical and physiological perspective" in a recent issue of FRBM.I It contains an evaluation of the long history on mechanisms of ascorbic acid regeneration that occur in animal tissues. There are two viewpoints: One group of investigators favors a significant role of enzyme- mediated conversion of the oxidized molecule (dehy- droascorbic acid, DHA); the traditional concept is that no such enzyme exists, and that "direct chemical re- duction" by glutathione is the physiologically im- portant reaction. The essence of the physiological as- pect of the review is that "GSH directly and nonenzy- matically reduces DHA to AA in simple solutions and in animal cells"; that is, nonenzymatic reduction is the important mechanism. Some points deserve comment.

A. The review suggests that in the several original articles that show evidence of enzyme-mediated reduction of DHA, 2-5 there is " a very low rate of activity, indeed and considerably below the nonen- zymatic rate under comparable conditions." How- ever, the data (e.g., Fig. 4 of ref. 6) indicate that the enzymatic rate consistently exceeds the nonen- zymatic rate.

B. Several laboratories have been unsuccessful in finding evidence of enzyme-mediated reduction of the oxidized form of ascorbic acid. However, there are numerous ways to destroy or mask the activity of an enzyme during processing of a tissue or assaying for activity.

Maellaro and coworkers 7 have confirmed with the use of rat liver many of our previous findings on kid-

ney, colon, brain, and eye tissue and have extended this work by purification and characterization of an enzyme with dehydroascorbate reductase activity. After three decades of controversy, evidence of a spe- cific glutathione-dependent DHA-reductase has been presented, and we congratulate the Italian investiga- tors. Furthermore, we predict that future research pa- pers will focus on this and possibly on additional re- lated enzymes that mediate the physiologically im- portant mode of ascorbate recycling.

Richard C. Rose Chicago Medical School North Chicago, IL 60064, USA

Ann M. Bode University of North Dakota School of Medicine Grand Forks, ND 58202, USA

REFERENCES

1. Winkler, B. S.; Orselli, S. M.; Rex, T. S. The redox couple between glutathione and ascorbic acid: A chemical and physio- logical perspective. Free Radic. Biol. Med. 17:333-349; 1994.

2. Choi, J.-L.; Rose, R. C. Regeneration of ascorbic acid by rat colon. Proc. Soc. Exp. Biol. Med. 190:369-378; 1989.

3. Rose, R. C. Renal metabolism of the oxidized form of ascorbic acid (dehydro-L-ascorbic acid). Am. J. Physiol. 256:F52-F56; 1989.

4. Bode, A. M.; Yavarow, C. R.; Fry, D. A.; Vargas, T. Enzymatic basis for altered ascorbic acid and dehydroascorbic acid levels in diabetes. Biochem. Biophys. Res. Commun. 191:1347-1353; 1993.

5. Rose, R. C.; Bode, A. M. Ocular ascorbate transport and metabo- lism. Comp. Biochem. Physiol. Part A. 100A:273-285; 1991.

6. Bode, A. M.; Green, E.; Yavarow, C. R.; Wheeldon, S. L.; Blo- ken, S.; Gomez, Y.; Rose, R. C. Ascorbic acid regeneration by bovine iris-ciliary body. Curr. Eye Res. 12:593-601; 1993.

7. Maellaro, E.; Del Bello, B.; Sugherini, L.; Comporti, M.; Casini, A. F. Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. Biochem. J. 301:471- 476; 1994.

REPLY TO DRS.

To the Editors:

We can understand the concern of Drs. Rose and Bode, since they believe that our article t does not acknowledge the existence of dehydroascorbate re- ductase. However, it was not our intent to dismiss its

R O S E A N D B O D E

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existence, but rather to downplay its physiological significance in mammalian tissues. The recent publi- cation of Maellaro et al. 2 subsequent to the submis- sion of our article supports Drs. Rose and Bode 's contention that there is reductase activity. Neverthe- less, the magnitude of this activity relative to that

956 Letters to the Editors

of the uncatalyzed rate is no more than 2 - 5 - f o l d higher. So, we concede, data being data, that reduc- tase activity is present in some mammal ian tissues, though we have not detected activity in retina, ciliary body, lens, and pigment epithelial cells.

We feel that Rose and Bode have allowed their focus on the existence of the enzyme to obscure their view of the more important picture regarding the inter- action between glutathione and ascorbic acid and their roles as cellular antioxidants. We attempted to bring a new perspective to the problem by analyzing the extent to which ascorbic acid could be regenerated following an oxidative challenge, a test particularly germane to its physiological role. Our results showed that retinal pigment epithelial cells are unable to regenerate ascorbic acid (see Fig. 13 of Winkler et al.l). Our results that do show reappearance of ascorbic acid in cells emphasize that this process takes place only upon supply of either dehydroascorbic acid or ascorbic acid in the medium following removal of the oxidative in- sult. These series of results clearly demonstrate that uptake of dehydroascorbic acid or ascorbic acid over- rides the enzymic and nonenzymic processes in the overall economy of ascorbic acid in the cell. It is this conclhsion that forms the cornerstone of what we had hoped readers would see as the exciting new contribu- tion in our article.

Because Rose and Bode bring up their 1993 article in Current Eye Research, 3 we are responding specifi- cally to comment " A . " We did not cite this article in our publication, because several of their observations made little sense to us. It is k n o w n 1'4'5 that the initial rate of the reaction between glutathione and dehy- droascorbic acid in a buffered solution increases up to 10-fold between pH = 7.0 and pH = 8.0. When an article contains data (see Fig. 3 in Bode et al. 3) that shows essentially the same nonenzymatic rate between pH = 7.0 and pH = 8.0, we become skeptical. An

additional concern is that Bode et alJ 3 run their experi- ment for 10 min and then stop the reaction. They as- sume that the reaction rate is linear over the entire l0 min, because they express their results as a rate, that is, pmole Asc acid/min. However, this assumption is incorrect, because we ~ and others 2'6 find that the reac- tion rate at 30°C trails off after several minutes: Mael- laro et al. 2 write (p. 472) " the reaction was linear at least up to 2 min at 30°C. ' ' Another concern is that Bode et al. 3 reported that the enzyme requires both glutathione and NADPH, but do not explain the need for two reductants. Moreover, this result also distin- guishes their enzyme from that of Maellaro et al., 2 which needed no NADPH.

Barry S. Winkler Stephen M. Orselli Tonia S. Rex Eye Research Institute Oakland University Rochester, MI, 48309, USA

REFERENCES

1. Winkler, B. S.; Orselli, S.; Rex, T. S. The redox couple between glutathione and ascorbic acid: A chemical and physiological per- spective. Free Radic. Biol. Med. 17:333-349; 1994.

2. Maellaro, E.; Dei Bello, B.; Sugherini, L.; Santucci, A.; Comporti, M.; Casini, A. F. Purification and characterization of glutathione- dependent dehydroaseorbate reduetase from rat liver. Biochem. J. 301:471-476; 1994.

3. Bode, A. M.; Green, E.; Yavarow, C. R.; Wheeldon, S. L.; Bol- ken, S.; Gomez, Y.; Rose, R. C. Ascorbic acid regeneration by bovine iris-ciliary body. Curr. Eye Res. 12:593-601; 1993.

4. Hughes, R. E. Reduction of dehydroascorbic acid by animal tis- sues. Nature 203:1068-1069; 1964.

5. Bigley, R.; Riddle, M.; Layman, D.; Stankova, L. Human cell dehydroascorbate reductase. Biochim. Biophys. Acta 659:15-22; 1981.

6. Wells, W. W.; Xu, D. P.; Yang, Y.; Rocque, P. A. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J. Biol. Chem. 265:15361-15364; 1990.