The metabolism and acid degradation of quercetin

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protein-cytochrome b complex contains some other form of the dehydrogenase or some “factor” required to link the flavoprotein to coenzyme Q. The following reaction scheme would account for the observations recorded above: cytochrome b succinic + flavoprotein

7 I

coenzyme Q

ACKNOWLEDGEMENTS

We wish to express our gratitude to Dr. D. E. Green for his suggestions and interest in this work, and we thank Sally Krueger for her valuable technical assistance. This work was supported in part by Division of Research Grants Graduate Training Grant 20-88 and National Heart Institute Grant H-458 both from the NIH, USPHS; NSF Grant G-3227; and AEC Contract AT(ll-l)-64, Project 4. Beef hearts were generously supplied by Oscar Mayer and Co., Madison, Wisconsin. REFERENCES

1. PAPPENHEIMER, A. M., JR., AND HENDEE, E. D., J. Biol. Chem. 180, 597 (1949). 2. LARA, F. J. S., Biochim. et Biophys. Acta 33, 565 (1959). 3. HUBSCHER, G., KIESE, M., AND NICOLAS, R., Biochem. 2. 326, 223 (1954). 4. J~RNEFELT, J., BASFORD, R. E., TISDALE, H. D., AND GREEN, D. E., Biochim. et Biophys. Actu 29, 123 (1958). 5. NYGAARD, A. P., Biochim. et Biophys. Actu 33, 517 (1959). 6. SINGER, T. P., KEARNEY, E. B., AND BERNATN, P., J. Biol. Chem. 223, 599 (1956). D. M. ZIEGLER K. A. DOEG’

Institute for Enzyme Research University of Wisconsin Madison, Wisconsin Received July 23, 1969

1 Postdoctoral trainee of the University search, Madison, Wisconsin. The Metabolism

of Wisconsin, Institute

and Acid Degradation

for Enzyme Re-

of Quercetin

Metabolic studies from this laboratory (1, 2) have shown that the flavonoid quercetin undergoes metabolic degradation giving rise to the following two-carbon side chain phenolic acids: 3,4-dihydroxyphenylacetic, m-hydroxyphenylacetic, and homovanillic acids. The chemical degradation of quercetin with alkali (3), on the other hand, results in cleavage of the heterocyclic ring to give the one-carbon side chain phenolic acid, 3,4-dihydroxybenxoic (protocatechuic) acid. This treatment with alkali also yields phloroglucinol and phloroglucinolcarboxylic acid, thus accounting for the other portion of the molecule. In the metabolic studies cited above, however, phloroglucinol, and phloroglucinolcarboxylic acid were not detected in the urine. Douglass (4) reported the formation of protocatechuic acid when quercetin was incubated with rat kidney homogenates. Recently, Wender and co-workers (5) reported the presence of protocatechuic acid, phloroglucinol, and phloroglucinol carboxylic acid in the gastrointestinal tract of rats given C-quercetin by stomachtube. A small amount of expired Cl402 was also found, but no data on urinary metabolites were reported. It is interesting to note that these authors experienced difficulty in the complete extraction of these compounds from the intestinal tract. To achieve

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complete recovery they resorted to autoclaving the samples in 0.1 N HCl for 10 min. at 15 lbs. psi. prior to extraction with ether. These authors were able to detect protocatechuic acid, phloroglucinolcarboxylic acid, and phloroglucinol in lyophilized, unautoclaved stomach and contents. In this note we present observations on the metabolism of C”-quercetin in the rat which confirm our previous findings with unlabeled quercetin. Observations on the chemical degradation of quercetin in acid media are also reported. Radioactive rutin was isolated according to described procedures (6) from the flowers of the yellow pansy, after the budding plants were allowed to fix CY402 for several days. The C4-rutin was crystallized with unlabeled carrier from hot water and recrystallized from alcohol. CP-quercetin was obtained by acid hydrolysis of the Cl4-rutin, and crystallized from acetone-water. Both C-rutin and CL4-quercetin were checked for purity by preparing radioautographs of two-dimensional chromatograms, using n-butanol, acetic acid, water, 5:1:1, in one direction and 20y0 KC1 in the other. The Cl”-quercetin (80 mg.) was given by stomach tube to a rat and the urine and expired COz were collected for 24 hr. The expired Cl92 was too highly diluted with unlabeled CO2 to permit measurement. An ether extract of the collected urine was chromatographed as described elsewhere (2) and a radioautograph was prepared. Four distinct spots on the radioautograph corresponded to 3,4-dihydroxyphenylacetic acid, m-hydroxyphenylacetic acid, and its glucuronide, and homovanillic acid on the chromatogram. There were no spots on either the radioautograph or chromatogram corresponding to the positions of protocatechuic acid, phloroglucinolcarboxylic acid, or phloroglucinol. If these compounds were present, their amounts must have been, therefore, very small. The finding of areas on the radioautograph corresponding to m-hydroxyphenylacetic acid as well as 3,4-dihydroxyphenylacetic acid confirms the previously reported phenomenon of dehydroxylation (2). Similarly, the presence of radioactive homovanillic acid confirms the 0-methylation of 3,4-dihydroxyphenylacetic acid (2). The difficulty experienced by Wender and co-workers (5) in the complete extraction of protocatechuic acid and phloroglucinol without prior autoclaving in 0.1 N HCl prompted us to subject samples of pure unlabeled quercetin to the following procedures: (1) 10 min. autoclaving in 0.1 N HCl at 30 lbs. p.s.i., (2) 20 min. autoclaving in 0.1 N HCl at 90 lbs. p.s.i., (3) incubation in 0.1 N HCl overnight at 37°C. and atmospheric pressure, (4) incubation in water overnight at 37°C. and atmospheric pressure. All these procedures except the last one resulted in the formation of quite small but chromatographically detectable amounts of protocatechuic acid, phloroglucinolcarboxylic acid, and phloroglucinol in the ether extracts of the treated samples. In our studies 80 mg. of quercetin were administered to a rat in contrast with the 4-10 mg. employed by Wender and associates. The failure to detect phloroglucinol, phloroglucinolcarboxylic acid, and protocatechuic acid in the urine indicates that acid degradation of quercetin in the stomach did not occur to a significant extent in our experiment. It is interesting to note that similar treatments in acid media of the flavonoids naringenin and hesperetin also resulted in degradation. In fact, the latter flavonoids were more vulnerable to such treatment than quercetin. The formation of protocatechuic acid, phloroglucinol, and phloroglucinolcarboxylic acid from quercetin in the presence of 0.1 N HCI suggests that the finding of these products in the gastrointestinal tract of rats as reported by Wender and coworkers may be due to chemical degradation rather than to metabolic processes. Since Cd-quercetin obtained by C40s fixation is evenly labeled (3), the phloro-

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glucinol moiety would be expected to contain approximately half of the radioactivity. The absence from the urine, in our experiments, of phloroglucinol and phloroglucinolcarboxylic acid after administration of Cl”-quercetin, while the other portion of the molecule was accounted for by radioautographic evidence of 3,4-dihydroxyphenylacetic, m-hydroxyphenylacetic, and homovanillic acids, suggests that the phloroglucinol moiety is completely degraded to CO2 . When phloroglucinolcarboxylic acid (100 mg.) was given to rats by stomach-tube, an appreciable portion was found in the urine 24 hr. later, either unchanged or as phloroglucinol; similarly, when phloroglucinol (100 mg.) was administered, a major portion was excreted unchanged. If quercetin undergoes a metabolic cleavage through the heterocyclic ring, it would therefore be expected that phloroglucinolcarboxylic acid and phloroglucinol would be found in the urine. It appears from these observations that the concept of a cleavage of the heterocyclic ring of quercetin to yield two primary fragments is not satisfactory. Experiments to be reported in a later communication indicate that, rather than a cleavage through the heterocyclic ring, quercetin undergoes degradation from the phloroglucinol end of the molecule in a manner that does not result in the intermediate formation of free phloroglucinolcarboxylic acid or phloroglucinol. The dihydroxyphenyl residue then gives the metabolites previously reported. It is pertinent to mention here also that oral administration of protocatechuic acid (100 mg.) to rats invariably resulted in the excretion of vanillic acid and vanilloylglycine, as well as protocatechuic acid itself (7). No evidence of vanillic acid was found on the chromatogram or radioautograph prepared from urine collected after administration of C14-quercetin. The above may be summarized as follows: (1) oral administration of C14-quercetin to a rat resulted in the urinary excretion of radioactive 3,4-dihydroxyphenylacetic acid, m-hydroxyphenylacetic acid and its glucuronide, and homovanillic acid, (2) no evidence was found for the presence of protocatechuic acid, phloroglucinolcarboxylic acid, or phloroglucinol, (3) quercetin undergoes chemical degradation in acid media to yield small amounts of protocatechuic acid, phloroglucinolcarboxylic acid, and phloroglucinol. REFERENCES 1. MURRAY, C. W., BOOTH, A. N., DEEDS, F., AND JONES, F. T., J. Am. Pharm. Assoc., Sci. Ed. 43, 361 (1954). 2. BOOTH, A. N., MURRAY, C. W., JONES, F. T., AND DEEDS, F., J. Biol. Chem. 333, 251 (1956). 3. WATKIN, J. E., UNDERHILL, E. W., AND NEISH, A. C., Can. J. Biochem. Physiol. 36, 229 (1957). 4. DOUGLASS, C. D., Federation Proc. 17, 213 (1958). 5. KALLIANOS, A. G., PETRAKIS, P. L., SHETLAR, M. R., AND WENDER, S. H., Arch. Biochem. Biophys. 81, 430 (1959). 6. GRIFFITH, J. R., KREWSON, C. F., AND NAGHSKI, J., “Rutin and Related Flavonoids,” p. 24. Mack Publishing Co., Easton, Penn. 7. DEEDS, F., BOOTH, A. N., AND JONES, F. T., J. Biol. Chem. 226, 615 (1957). M. S. MASRI Western Regional Research Laboratory A. N. BOOTH Western Utilization Research and F. DEEDS Development Division Agricultural Research Service U. S. Department of Agriculture Albany, California Received July 30, 1959