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O-methylation in vitro of dihydroxy- and trihydroxy-phenolic compounds by liver slices
BIOCHIMICA ET BIOPHYSICA ACTA
495
BBA 3825
O-METHYLATION IN
VITRO
TRIHYDROXY-PHENOLIC
OF D I H Y D R O X Y - AND
COMPOUNDS BY L I V E R SLICES
M. S. MASRI, A. N. BOOTH, AND F. DEEDS Western Regional Research Laboratory*, Albauy, Calif. (U.S.A.)
(Received June iSth, 1962)
SUMMARY Several instances of O-methylation of dihydroxy- and trihydroxy-phenolic acids are shown in vitro with surviving rat or rabbit liver slices. Instances are shown where O-methylation affects the m-hydroxyl group specifically, the p-hydroxyl group specifically, or either one of the m- or p-hydroxyl groups of the same phenolic acid indifferently. Some factors which influence the course of O-methylation are discussed.
INTRODUCTION Studies on the metabolic fate of rutin and quercetin in experimental animals by BOOTH et al. 1,~ led to the discovery that phenolic compounds with adjacent hydroxyl groups undergo O-methylation. This reaction, which may be regarded as a mode of detoxication, has been repeatedly demonstrated with a number of compounds by DEEDs et al3., BOOTH et al. 4, MASRI et a l ) and BOOTH etal. ~. O-Methylation has been confirmed by SHAWet ald, ARMSTRONGet ~l. 8, AXELRODgt al. 9, PELLERIN 6~al. I°, and WATANABE11. AXELROD et al. 9 showed that the responsible enzyme is in the supernatant fraction of mammalian liver after homogenization with isotonic potassium chloride. Our results with the intact animal showed that in a series of 2,4-dihydroxyphenolic compounds methylation usually affected the m-hydroxyl group. However, in the case of 3,4dihydroxycinnamic (caffeic) acid BOOTH et al. 4 found a small amount of 3-hydroxy4-methoxycinnamic (isoferulic) acid, indicative of p-O-methylation, in addition to the major metabolite 3-methoxy-4-hydroxycinnamic (ferulic) acid. In studies on the metabolism of 3,4,5-trihydroxybenzoic (gallic) acid BOOTH et al. ~ showed that p-Omethylation occurred to the exclusion of m-O-methylation. SENOH et al. ~2 and DALY et al. ~3 also demonstrated p-O-methylation of other compounds. The present report includes further examples of p-O-methylation with compomlds which have not been shown previously to undergo this reaction. Compounds lacking an o r t h o - d i h y d r o x y configuration are also included. The results of these studies i s vitro with liver slices afford some conclusions relating structure of the polyhydroxyphenolic substrates and their metabolic disposition toward O-methylation. They also permit conclusions regarding the selectivity of m- or p-O-methylation in relation to structure of the substrate. * A laboratory of the V~'estern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. Biochim. Biophys. Aeta, 65 (I962) 495 505
496
M. S. MASRI, A. N. BOOTH, F. D E E D s EXPERIMENTAL
Most of the phenolic acids studied were obtained as described by BOOTH et al. ~, 4,6 and DEEDS et al. ~. Others were obtained from commercial sources. The purity of all compounds was checked chromatographically. Male or female albino rats from a colony maintained in this laboratory and weighing 15o-2oo g each were fed ad libitum a diet previously described by MASRI et al. 14. The rats were anesthetized lightly with ether, sacrificed and their livers excised quickly and placed in a bicarbonate buffer described by KREBS et al. 15. Liver slices approx. 0. 5 mm thick were prepared free-hand with a sharp razor blade. Approx. I g of slices was incubated with shaking at 37 ° in a buffered medium. The medium consisted of IO ml of the bicarbonate buffer previously equilibrated with O2-CO2, (95 : 5), to which were added 20 mg glucose, I mg each of choline chloride, nicotinamide, pyridoxine hydrochloride and ascorbic acid. The substrate consisted of 2 mg of the phenolic compound under investigation. Control flasks were employed in which either the substrate or the liver was withheld. Incubation was continued for about 4 h. In certain experiments rabbit liver was used instead of rat liver. A single rat liver usually served to furnish slices for 4-5 incubation flasks. Each substrate was tested on different occasions (with different rat livers) more than once. In order to check and compare the ability of different livers to perform O-methylation, usually a flask was employed in which a portion of the particular liver was incubated with caffeic acid as a reference substrate. At the end of the incubation period, the slices were mashed to a paste in a mortar (facilitated with solid NaC1). The medium and the paste were combined, saturated with solid NaC1, acidified to pH 2-3 with HCI, filtered with Celite and the filtrate extracted with ether. The total ether extract or an aliquot thereof was chromatographed two-dimensionally using chloroform-acetic acid-water (2:1 : i, lower phase) in one direction and 2o % KC1 in the second direction. The chromatographic procedures for the identification of the metabolites encountered in the present work have been described by BOOTH et al. 2, 4,6 and DEEDs et al. a. The RE values and other chromatographic data for the substrates used and their metabolites are shown in Table I. The amounts of metabolites formed were estimated by visual comparison of the size and color intensity of the spots formed on the sprayed chromatograms with known amounts of the authentic compounds. RESULTS
The patterns of O-methylation revealed by chromatographic examination of tim ether extracts of the incubation flasks with the various substrates are described below. With the substrates which were methylated readily, such as protocatechuic, 3,4dihydroxyphenylacetic, caffeic, gallic and 2,3,4-trihydroxybenzoic acids, the total amount of free methylated metabolites (including both isomers when formed) was of the order of 5O-lOO/~g per incubation flask. The sensitivity of the chromatographic technique is capable of detecting as little as 2 #g of a given phenolic acid. 3,4-Dihydroxybenzoic (protocatechuic) acid
Incubation with this substrate gave rise to 3-methoxy-4-hydroxybenzoic (vanillic) acid and its glycine conjugate. In some experiments small amounts of 3-hydroxy-4Biochim. Biophys..4cta, ~5 (1(702) 495 505
O-METHYLATION OF POLYHYDROXYPHENOLIC COMPOUNDS
497
TABLE I CHROMATOGRAPHIC
BEHAVIOR
RF • Compounds
CHCI3 acetic - H~O
20 % KCl
OF
PHENOLIC
COMPOUNDS
Appearance under ultraviolet
Color with diazotized sulfanilic acid
Protocatechuic
0.09
o.4I
Deep blue
Pink-tan, darkens
Vanillic
0.78
o.42
Deep blue, phosphoresces
Orange
Isovanillic Vanilloylglycine
0.7o o. 12
0.43 o. 64
Deep blue Deep blue, phosphoresces
Orange Red-Orange
Isovanilloylglycine 3,4 - D i h y d r o x y p h e n y l a c e t i c Homovanillic Isohomovanillic
o. I3 o.o8 o. 78 o. 71 o.i i
o.65 o.75 o.83 o. 84 o.I6
Blue Absorbs Absorbs Absorbs Blue
Red-orange Red, d a r k e n s Purple-red Orange Gray-tan, darkens
trans-Ferulic**
o. 18 o.81
0.62 o.2o
Absorbs Blue
Red, d a r k e n s Blue-violet, fades
trans-Isoferulic**
o.76
o.17
Gray-blue
T a n - o r a n g e , fades
trans-Feruloylglycine
o. 14
0.27
L i g h t blue
Blue-violet, fades
trans-Isoferuloylglycine
o.12
o.25
L i g h t blue
Pink-orange, fades
Dihydroferulic Dihydroisoferulic Gallic acid 4-O-Methylgallic 3-0-Methylgallic 3,4-Dimethoxy-5hydroxybenzoic Syringic acid 2,3,4 -Trih y d r o x y b e n z o i c 3-Methoxy-2,4dihydroxybenzoic c~-Resoreylic
0.9o 0.9o o.o2 o. 16 0.20
o.62 o.64 0.45 0.56 0.43
Absorbs Absorbs Deep blue Deep blue Deep blue
Purple-red Orange Green, d a r k e n s Yellow-orange Reddish, d a r k e n s
o.76 0.84 o. 13
0.58 o.4o o.41
Deep blue Deep blue Blue
Yellow-orange Red Darkens
0.65 0.04
o. 55 o.43
Blue Deep blue
Yellow-orange Yellow-orange, periphery reddens
/~-Resorcylic
0.32
0. 41
Blue
Yellow-orange, periphery darkens
7-Resorcylic
0.20 streaks
0.43
Absorbs
Yellow-orange, periphery darkens
2,3-Dihydroxybenzoic
0. 51
0.46
L i g h t blue
Pink-tan, darkens
Pyrogallol
o. 14
o. 71
Absorbs
Darkens
I-O-Methylpyrogallol
o.8 t
o.61
Absorbs
Darkens
2-O-Methylpyrogallol
o.6J
0-79
Absorbs
Yellow-orange
trans-Caffeic
Dihydrocaffeic
* A s c e n d i n g c h r o m a t o g r a p h y on W h a t m a n No. i filter paper. T h e d i s t a n c e t r a v e l e d was m e a s u r e d to t h e c e n t e r of t h e s p o t of t h e c o m p o u n d . ** More s a t i s f a c t o r y s e p a r a t i o n of ferulic a n d isoferulic acids c a n be obtained, u s i n g t h e a b o v e c h l o r o f o r m s o l v e n t in one d i m e n s i o n a n d isopropyl a l c o h o l - c o n c e n t r a t e d a m m o n i u m h y d r o x i d e w a t e r (4 : I : i) in t h e second dimension. B i o c h i m . B i o p h y s . ,4cta, 65 (1962) 495 5c5
498
M.S. MASRI, A. N. BOOTH, F. DEEDs
methoxybenzoic (isovanillic) acid were also detected. The methylation of the phydroxyl group of this substrate (though occurring only to a small extent) has not been reported previously. Incubation with vanillic acid or isovanillic acid resulted in the formation of the corresponding glycine conjugate. The results with protocatechuic acid indicate preferential methylation of the m-hydroxyl group (m-O-methylation).
3,4-Dihydroxyphenylacetic acid This substrate gave rise to 3-methoxy-4-hydroxyphenylacetic (homovanillic) acid mostly, and, to a lesser extent but consistently, 3-hydroxy-4-methoxyphenylacetic (isohomovanillic) acid. In other words, preferential methylation of the mhydroxyl group of this substrate took place. The methylation of the p-hydroxyl group of this substrate has not been reported previously. Although the RF values of homovanillic and isohomovanillic acids are close with the solvents used, the two acids produce different colors after spraying with diazotized sulfanilic acid and sodium carbonate (Table I).
3,4-Dihydroxycinnamic (caffeic) and 3,4-dihydroxyphenvlpropionic (dihydrocaffeic) acids When either of these substrates was incubated with liver slices, the following methoxy derivatives were obtained: trans-3-methoxy-4-hydroxycinnamic (ferulic), dihydroferulic, trans-3-hydroxy-4-methoxycinnamic (isoferulic) and vanillic acids and feruloylglycine.The interconversion of caffeic and dihydrocaffeic acids occurred readily. The amount of isoferulic acid relative to that of ferulic acid formed from caffeic or dihydrocaffeic acids was appreciable, clearly indicating that p- as well as m-Omethylation took place readily with these substrate~,. Again, although the RF values of ferulic and isoferulic acids are close with the solvents employed, the two acids can be differentiated readily by the contrasting colors they produce as well as their behavior under ultraviolet light (Table I). When ferulic acid was used as a substrate, it gave rise to feruloylglycine, dihvdr-ferulic acid, vanillic acid and vanilloylglycine. Similar results were obtained with isoferulic acid ; here, the corresponding isomers of the metabolites of ferulic acid xverobtained,
3,4,5-Trihydroxvbenzoic (gallic) and 2,3,4-trihydvo.zybenzoic acids Gallic acid gave only 4-0-methyl gallic acid (i.e. p-O-methylation selectively) while 2,3,4-trihydroxybenzoic acid gave only 3-methoxy-2,4-dihydroxybenzoic acid (i.e. m-O-methylation selectively). Similar results were also obtained when rabbit liver slices were used instead of rat liver slices.
3-O-Methylgallic acid Here, m- as well as p-O-methylation occurred as evidenced b y the formation o two dimethoxy isomers, namely, 3,4-dimethoxy-5-hydroxybenzoic and 3,5-dimethoxy4-hydroxybenzoic (syringic) acids. Again, similar results were obtained using rabbit liver slices.
Other acids No methylation was detected using 4-O-methylgallic or 3-methoxy-2,4dihydroxybenzoie acids. When either ~-,/3- or y-resorcylic (3,5-, 2,4-, or 2,6-dihydroxy-
Biochir~z. Bioph3,s. Acta, 05 (~962) 495 5~5
O-METHYLATION OF POLYHYDROXYPHENOLIC COMPOUNDS
499
benzoic) acids were used as substrates, no methoxy derivatives were detected. Similarly, no methoxy derivatives were found with 2,3-dihydroxybenzoic acid, although with this acid a small amount of 2-hydroxy-3-methoxybenzoic acid was detected in the urine in an in vivo experiment. Pyrogallol and z-O-methylpyrogallol When either of these substrates were incubated with liver slices, methylated derivatives could not be detected. Instead, conjugates of these substrates were formed, having low Rp values in the direction of the chloroform phase and high RE values in the direction of the aqueous phase, forming a yellow color after spraying with diazotized sulfanilic acid-sodium carbonate, and liberating the original substrates after hydrolysis. When catechol was used as a substrate, appreciable conjugation also took place, but since we could not detect known guaiacol, the monomethyl ether of catechol, b y our chromatographic procedure, its formation cannot be excluded. When portions of the same liver used in these experiments with phenols were incubated separately with gallic acid or protocatechuic acid, methylation of these latter substrates could be detected readily. DISCUSSION Eight of the 14 compounds investigated gave no evidence of O-methylation. They were 4-0-methylgallic acid, 3-methoxy-2,4-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, pyrogallol and I-0-methylpyrogallol. Of the three 3,4-dihydroxy acids Omethylation affected the m-hydroxyl group preferentially for 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid, the preference being greater in the former. In the case of 3,4-dihydroxycinnamic acid the m- and p-hydroxyl groups were equally affected. Results with these compounds, differing only in length of the side chain, suggest that increasing length of the side chain increases the possibility of p - o methylation. Comparison of the present results in vitro with protocatechuic, 3,4-dihydroxyphenylacetic, and caffeic acids with previous findings in the intact animal with the same acids reveals two important distinctions. First, p-O-methylation occurred to a greater extent in vitro with liver slices than was observed in the intact animal. Second, although the reaction of dehydroxylation (affecting the p-hydroxyl group) had been amply demonstrated and confirmed in the intact animal by BOOTH et al. 12, DEEDs et al. a, MASRI et al. a, WATANABE 11, BOOTH et al. 16, SHAW et a l Y and TOMPSETT 18, attempts to demonstrate this reaction with liver slices were not successful. That dehydroxylation is due to tissue enzymes, however, is indicated by the finding that it occurred when caffeic acid was given by injection*. It is interesting to note here that in the intact animal dehydroxylation of caffeie acid with a 3-carbon side chain occurred more abundantly than dehydroxylation of protocatechuie acid with a 1-carbon side chain 3. This is to be compared with the finding in the present study that p-O-methylation was similarly influenced by length of the side chain. The pattern of O-methylation obtained in vitro with gallic, 2,3,4-trihydroxybenzoic and 3-O-methylgallic acids is fully in agreement with the results previously reported using the intact animal 6. Thus, with the expenditure of a single methyl Biochim. Biophys. Ac/a, 65 (I962) 495 505
500
M . S . MASRI, A. N. BOOTH, F. D E E D s
group, the 2 potential ortho-dihydroxy configurations of gallic acid or 2,3,4-trihydroxybenzoic acid were simultaneously abolished by selective p- or m-O-methylation respectively. In contrast, with 3-0-methylgallic acid, which has but a single orthodihydroxy configuration, either m- or p-O-methylation occurred. With 4-0-methylgallic and 3-methoxy-2,4-dihydroxybenzoic acids, neither of which has an orthodihydroxy configuration, further O-methylation of these substrates was not observed. The a-, fl- and y-isomers of resorcylic acid which lack an ortho-dihydroxy group failed to yield methoxy derivatives in vitro. O-Methylation of the ortho-dihydroxy compound, 2,3-dihydroxybenzoic acid, also failed to occur in vitro, although a slight amount of O-methylation occurred in vivo (unpublished data). The results obtained with the catecholic phenols, pyrogallol and 1-0-methylpyrogallol were dissimilar to those obtained with the catecholic acids : with the phenols conjugation rather than methylation was the evident reaction in vitro with liver slices. The formation of conjugates indicated the liver slices were surviving in the presence of these phenols. These results are consistent with our observation that 50 mg of pyrogallol administered to a rat b y stomach tube resulted in the urinary excretion of large amounts of conjugates, and a trace of 2-0-methylpyrogallol. Similarly, conjugates but no 2,3-dimethoxyphenol were excreted in the urine following administration b y stomach tube of IOO mg of I-0-methylpyrogallol. In contrast ARCHER 6t al. 19 found incubation of pyrogallol with catechol 0-methyl transferase yielded 1-0-methyl pyrogallol and 2,3-dimethoxyphenol. Although pyrogallol is subject to O-methylation in this isolated enzyme system, it does not appear that the same metabolic pathway represents the manner in which the intact organism handles this compound. In this respect, the liver-slice technique, by virtue of its greater degree of integration, suffers less from the rather altificial nature of the technique in vitro. The difference in results obtained by incubation with surviving liver slices, and with isolated O-methyl transferase, as well as differences reported in this paper between the results of the liver-slice technique and those obtained with the intact animal emphasize the hazards of transferring results in vitro to the intact animal. REFERENCES N. BOOTH, C. W. MURRAY, F. D E E D s AND F. T. JONES, Federation Proc., 14 (1955) 32 E. N. BOOTH, C. W T. MURRAY, F. T. JONES AND F. D E E D s , J. Biol. Chem., 223 (1956) 251. D E E D s , A. N. BOOTH AND F. T. JONES, J. Biol. Chem., 225 (1957) 615. N. BOOTH, O. H . EMERSON, F. T. JONES AND V. D E E D s , J. Biol. Chem., 229 (1957) .51. S. MASRI, A. N. BOOTH AND F. D E E D s , Arch. Biochem. Biophys., 85 (1959) 284. N. BOOTH, M. S. MASRI, D. J. ROBBINS, O. H . EMERSON, F. T. JONES AND F. D E E D s , J. Biol. Chem., 234 (1959) 3o14. 7 K. N. F. SHAW, A. McMILLAN AND M. D. ARMSTRONG, J . Biol. Chem., 226 (t957) 255. s M. D. ARMSTRONG, A. McMILLAN AND K . ~N. F. SHAW, Biochim. Biophys. Acta, 25 (1957) 422. 9 j . AXELROD AND R . TOMCHICK, J. Biol. Chem., 233 (1958) 7o2. lO j . PELLERIN AND A. D ' I o R I O , Can. J. Biochem. Physiol., 36 (1958) 491. 11 H . WATANABE, Bull. Agr. Chem. Soc. Japan, 23 (1959) 268. 12 S. SENOH, J. DALY, J. AXELROD AND B. WITKOP, J. Am. Chem. Soc., 81 (19.59) 624o. la j . W . DALY, J. AXELROD AND B. ~VITKOP, J . Biol Chem., 235 (196o) 1155. la M. S. MASRI AND F. D E E D s , Proc. Soc. Exptl. Biol. Med., 99 (1958) 707 . 15 H. A. KREBS AND K. HENSELEIT, I . physiol. Chem. Hoppe-Seyler's, 21o (I932) 33. 1~ A. N. BOOTH, F. T. JONES AND V. D E E D s , J. Biol. Chem., 23o (19.58) 6 6 t . 17 K. N. F. SHAW AND J. TREVARTHEN, Nature, t82 (1958) 79718 S. L. TOMPSETT, J. Pharm. and Pharmacol., I I (1959) 32. 19 S. ARCHER, A. ARNOLD, R. K. KULLNIG AND D. \ ¥ . \¥YLIE, Arch. Biochem. BiophJ's., '~7 (I96O) 153. 1 A. 2 A. 3 F. 4 A. 5 M. 8 A.
Biochim. Biophy,~. ~4cta. (~5 ( 106- J 495 5o5
495
BBA 3825
O-METHYLATION IN
VITRO
TRIHYDROXY-PHENOLIC
OF D I H Y D R O X Y - AND
COMPOUNDS BY L I V E R SLICES
M. S. MASRI, A. N. BOOTH, AND F. DEEDS Western Regional Research Laboratory*, Albauy, Calif. (U.S.A.)
(Received June iSth, 1962)
SUMMARY Several instances of O-methylation of dihydroxy- and trihydroxy-phenolic acids are shown in vitro with surviving rat or rabbit liver slices. Instances are shown where O-methylation affects the m-hydroxyl group specifically, the p-hydroxyl group specifically, or either one of the m- or p-hydroxyl groups of the same phenolic acid indifferently. Some factors which influence the course of O-methylation are discussed.
INTRODUCTION Studies on the metabolic fate of rutin and quercetin in experimental animals by BOOTH et al. 1,~ led to the discovery that phenolic compounds with adjacent hydroxyl groups undergo O-methylation. This reaction, which may be regarded as a mode of detoxication, has been repeatedly demonstrated with a number of compounds by DEEDs et al3., BOOTH et al. 4, MASRI et a l ) and BOOTH etal. ~. O-Methylation has been confirmed by SHAWet ald, ARMSTRONGet ~l. 8, AXELRODgt al. 9, PELLERIN 6~al. I°, and WATANABE11. AXELROD et al. 9 showed that the responsible enzyme is in the supernatant fraction of mammalian liver after homogenization with isotonic potassium chloride. Our results with the intact animal showed that in a series of 2,4-dihydroxyphenolic compounds methylation usually affected the m-hydroxyl group. However, in the case of 3,4dihydroxycinnamic (caffeic) acid BOOTH et al. 4 found a small amount of 3-hydroxy4-methoxycinnamic (isoferulic) acid, indicative of p-O-methylation, in addition to the major metabolite 3-methoxy-4-hydroxycinnamic (ferulic) acid. In studies on the metabolism of 3,4,5-trihydroxybenzoic (gallic) acid BOOTH et al. ~ showed that p-Omethylation occurred to the exclusion of m-O-methylation. SENOH et al. ~2 and DALY et al. ~3 also demonstrated p-O-methylation of other compounds. The present report includes further examples of p-O-methylation with compomlds which have not been shown previously to undergo this reaction. Compounds lacking an o r t h o - d i h y d r o x y configuration are also included. The results of these studies i s vitro with liver slices afford some conclusions relating structure of the polyhydroxyphenolic substrates and their metabolic disposition toward O-methylation. They also permit conclusions regarding the selectivity of m- or p-O-methylation in relation to structure of the substrate. * A laboratory of the V~'estern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. Biochim. Biophys. Aeta, 65 (I962) 495 505
496
M. S. MASRI, A. N. BOOTH, F. D E E D s EXPERIMENTAL
Most of the phenolic acids studied were obtained as described by BOOTH et al. ~, 4,6 and DEEDS et al. ~. Others were obtained from commercial sources. The purity of all compounds was checked chromatographically. Male or female albino rats from a colony maintained in this laboratory and weighing 15o-2oo g each were fed ad libitum a diet previously described by MASRI et al. 14. The rats were anesthetized lightly with ether, sacrificed and their livers excised quickly and placed in a bicarbonate buffer described by KREBS et al. 15. Liver slices approx. 0. 5 mm thick were prepared free-hand with a sharp razor blade. Approx. I g of slices was incubated with shaking at 37 ° in a buffered medium. The medium consisted of IO ml of the bicarbonate buffer previously equilibrated with O2-CO2, (95 : 5), to which were added 20 mg glucose, I mg each of choline chloride, nicotinamide, pyridoxine hydrochloride and ascorbic acid. The substrate consisted of 2 mg of the phenolic compound under investigation. Control flasks were employed in which either the substrate or the liver was withheld. Incubation was continued for about 4 h. In certain experiments rabbit liver was used instead of rat liver. A single rat liver usually served to furnish slices for 4-5 incubation flasks. Each substrate was tested on different occasions (with different rat livers) more than once. In order to check and compare the ability of different livers to perform O-methylation, usually a flask was employed in which a portion of the particular liver was incubated with caffeic acid as a reference substrate. At the end of the incubation period, the slices were mashed to a paste in a mortar (facilitated with solid NaC1). The medium and the paste were combined, saturated with solid NaC1, acidified to pH 2-3 with HCI, filtered with Celite and the filtrate extracted with ether. The total ether extract or an aliquot thereof was chromatographed two-dimensionally using chloroform-acetic acid-water (2:1 : i, lower phase) in one direction and 2o % KC1 in the second direction. The chromatographic procedures for the identification of the metabolites encountered in the present work have been described by BOOTH et al. 2, 4,6 and DEEDs et al. a. The RE values and other chromatographic data for the substrates used and their metabolites are shown in Table I. The amounts of metabolites formed were estimated by visual comparison of the size and color intensity of the spots formed on the sprayed chromatograms with known amounts of the authentic compounds. RESULTS
The patterns of O-methylation revealed by chromatographic examination of tim ether extracts of the incubation flasks with the various substrates are described below. With the substrates which were methylated readily, such as protocatechuic, 3,4dihydroxyphenylacetic, caffeic, gallic and 2,3,4-trihydroxybenzoic acids, the total amount of free methylated metabolites (including both isomers when formed) was of the order of 5O-lOO/~g per incubation flask. The sensitivity of the chromatographic technique is capable of detecting as little as 2 #g of a given phenolic acid. 3,4-Dihydroxybenzoic (protocatechuic) acid
Incubation with this substrate gave rise to 3-methoxy-4-hydroxybenzoic (vanillic) acid and its glycine conjugate. In some experiments small amounts of 3-hydroxy-4Biochim. Biophys..4cta, ~5 (1(702) 495 505
O-METHYLATION OF POLYHYDROXYPHENOLIC COMPOUNDS
497
TABLE I CHROMATOGRAPHIC
BEHAVIOR
RF • Compounds
CHCI3 acetic - H~O
20 % KCl
OF
PHENOLIC
COMPOUNDS
Appearance under ultraviolet
Color with diazotized sulfanilic acid
Protocatechuic
0.09
o.4I
Deep blue
Pink-tan, darkens
Vanillic
0.78
o.42
Deep blue, phosphoresces
Orange
Isovanillic Vanilloylglycine
0.7o o. 12
0.43 o. 64
Deep blue Deep blue, phosphoresces
Orange Red-Orange
Isovanilloylglycine 3,4 - D i h y d r o x y p h e n y l a c e t i c Homovanillic Isohomovanillic
o. I3 o.o8 o. 78 o. 71 o.i i
o.65 o.75 o.83 o. 84 o.I6
Blue Absorbs Absorbs Absorbs Blue
Red-orange Red, d a r k e n s Purple-red Orange Gray-tan, darkens
trans-Ferulic**
o. 18 o.81
0.62 o.2o
Absorbs Blue
Red, d a r k e n s Blue-violet, fades
trans-Isoferulic**
o.76
o.17
Gray-blue
T a n - o r a n g e , fades
trans-Feruloylglycine
o. 14
0.27
L i g h t blue
Blue-violet, fades
trans-Isoferuloylglycine
o.12
o.25
L i g h t blue
Pink-orange, fades
Dihydroferulic Dihydroisoferulic Gallic acid 4-O-Methylgallic 3-0-Methylgallic 3,4-Dimethoxy-5hydroxybenzoic Syringic acid 2,3,4 -Trih y d r o x y b e n z o i c 3-Methoxy-2,4dihydroxybenzoic c~-Resoreylic
0.9o 0.9o o.o2 o. 16 0.20
o.62 o.64 0.45 0.56 0.43
Absorbs Absorbs Deep blue Deep blue Deep blue
Purple-red Orange Green, d a r k e n s Yellow-orange Reddish, d a r k e n s
o.76 0.84 o. 13
0.58 o.4o o.41
Deep blue Deep blue Blue
Yellow-orange Red Darkens
0.65 0.04
o. 55 o.43
Blue Deep blue
Yellow-orange Yellow-orange, periphery reddens
/~-Resorcylic
0.32
0. 41
Blue
Yellow-orange, periphery darkens
7-Resorcylic
0.20 streaks
0.43
Absorbs
Yellow-orange, periphery darkens
2,3-Dihydroxybenzoic
0. 51
0.46
L i g h t blue
Pink-tan, darkens
Pyrogallol
o. 14
o. 71
Absorbs
Darkens
I-O-Methylpyrogallol
o.8 t
o.61
Absorbs
Darkens
2-O-Methylpyrogallol
o.6J
0-79
Absorbs
Yellow-orange
trans-Caffeic
Dihydrocaffeic
* A s c e n d i n g c h r o m a t o g r a p h y on W h a t m a n No. i filter paper. T h e d i s t a n c e t r a v e l e d was m e a s u r e d to t h e c e n t e r of t h e s p o t of t h e c o m p o u n d . ** More s a t i s f a c t o r y s e p a r a t i o n of ferulic a n d isoferulic acids c a n be obtained, u s i n g t h e a b o v e c h l o r o f o r m s o l v e n t in one d i m e n s i o n a n d isopropyl a l c o h o l - c o n c e n t r a t e d a m m o n i u m h y d r o x i d e w a t e r (4 : I : i) in t h e second dimension. B i o c h i m . B i o p h y s . ,4cta, 65 (1962) 495 5c5
498
M.S. MASRI, A. N. BOOTH, F. DEEDs
methoxybenzoic (isovanillic) acid were also detected. The methylation of the phydroxyl group of this substrate (though occurring only to a small extent) has not been reported previously. Incubation with vanillic acid or isovanillic acid resulted in the formation of the corresponding glycine conjugate. The results with protocatechuic acid indicate preferential methylation of the m-hydroxyl group (m-O-methylation).
3,4-Dihydroxyphenylacetic acid This substrate gave rise to 3-methoxy-4-hydroxyphenylacetic (homovanillic) acid mostly, and, to a lesser extent but consistently, 3-hydroxy-4-methoxyphenylacetic (isohomovanillic) acid. In other words, preferential methylation of the mhydroxyl group of this substrate took place. The methylation of the p-hydroxyl group of this substrate has not been reported previously. Although the RF values of homovanillic and isohomovanillic acids are close with the solvents used, the two acids produce different colors after spraying with diazotized sulfanilic acid and sodium carbonate (Table I).
3,4-Dihydroxycinnamic (caffeic) and 3,4-dihydroxyphenvlpropionic (dihydrocaffeic) acids When either of these substrates was incubated with liver slices, the following methoxy derivatives were obtained: trans-3-methoxy-4-hydroxycinnamic (ferulic), dihydroferulic, trans-3-hydroxy-4-methoxycinnamic (isoferulic) and vanillic acids and feruloylglycine.The interconversion of caffeic and dihydrocaffeic acids occurred readily. The amount of isoferulic acid relative to that of ferulic acid formed from caffeic or dihydrocaffeic acids was appreciable, clearly indicating that p- as well as m-Omethylation took place readily with these substrate~,. Again, although the RF values of ferulic and isoferulic acids are close with the solvents employed, the two acids can be differentiated readily by the contrasting colors they produce as well as their behavior under ultraviolet light (Table I). When ferulic acid was used as a substrate, it gave rise to feruloylglycine, dihvdr-ferulic acid, vanillic acid and vanilloylglycine. Similar results were obtained with isoferulic acid ; here, the corresponding isomers of the metabolites of ferulic acid xverobtained,
3,4,5-Trihydroxvbenzoic (gallic) and 2,3,4-trihydvo.zybenzoic acids Gallic acid gave only 4-0-methyl gallic acid (i.e. p-O-methylation selectively) while 2,3,4-trihydroxybenzoic acid gave only 3-methoxy-2,4-dihydroxybenzoic acid (i.e. m-O-methylation selectively). Similar results were also obtained when rabbit liver slices were used instead of rat liver slices.
3-O-Methylgallic acid Here, m- as well as p-O-methylation occurred as evidenced b y the formation o two dimethoxy isomers, namely, 3,4-dimethoxy-5-hydroxybenzoic and 3,5-dimethoxy4-hydroxybenzoic (syringic) acids. Again, similar results were obtained using rabbit liver slices.
Other acids No methylation was detected using 4-O-methylgallic or 3-methoxy-2,4dihydroxybenzoie acids. When either ~-,/3- or y-resorcylic (3,5-, 2,4-, or 2,6-dihydroxy-
Biochir~z. Bioph3,s. Acta, 05 (~962) 495 5~5
O-METHYLATION OF POLYHYDROXYPHENOLIC COMPOUNDS
499
benzoic) acids were used as substrates, no methoxy derivatives were detected. Similarly, no methoxy derivatives were found with 2,3-dihydroxybenzoic acid, although with this acid a small amount of 2-hydroxy-3-methoxybenzoic acid was detected in the urine in an in vivo experiment. Pyrogallol and z-O-methylpyrogallol When either of these substrates were incubated with liver slices, methylated derivatives could not be detected. Instead, conjugates of these substrates were formed, having low Rp values in the direction of the chloroform phase and high RE values in the direction of the aqueous phase, forming a yellow color after spraying with diazotized sulfanilic acid-sodium carbonate, and liberating the original substrates after hydrolysis. When catechol was used as a substrate, appreciable conjugation also took place, but since we could not detect known guaiacol, the monomethyl ether of catechol, b y our chromatographic procedure, its formation cannot be excluded. When portions of the same liver used in these experiments with phenols were incubated separately with gallic acid or protocatechuic acid, methylation of these latter substrates could be detected readily. DISCUSSION Eight of the 14 compounds investigated gave no evidence of O-methylation. They were 4-0-methylgallic acid, 3-methoxy-2,4-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, pyrogallol and I-0-methylpyrogallol. Of the three 3,4-dihydroxy acids Omethylation affected the m-hydroxyl group preferentially for 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid, the preference being greater in the former. In the case of 3,4-dihydroxycinnamic acid the m- and p-hydroxyl groups were equally affected. Results with these compounds, differing only in length of the side chain, suggest that increasing length of the side chain increases the possibility of p - o methylation. Comparison of the present results in vitro with protocatechuic, 3,4-dihydroxyphenylacetic, and caffeic acids with previous findings in the intact animal with the same acids reveals two important distinctions. First, p-O-methylation occurred to a greater extent in vitro with liver slices than was observed in the intact animal. Second, although the reaction of dehydroxylation (affecting the p-hydroxyl group) had been amply demonstrated and confirmed in the intact animal by BOOTH et al. 12, DEEDs et al. a, MASRI et al. a, WATANABE 11, BOOTH et al. 16, SHAW et a l Y and TOMPSETT 18, attempts to demonstrate this reaction with liver slices were not successful. That dehydroxylation is due to tissue enzymes, however, is indicated by the finding that it occurred when caffeic acid was given by injection*. It is interesting to note here that in the intact animal dehydroxylation of caffeie acid with a 3-carbon side chain occurred more abundantly than dehydroxylation of protocatechuie acid with a 1-carbon side chain 3. This is to be compared with the finding in the present study that p-O-methylation was similarly influenced by length of the side chain. The pattern of O-methylation obtained in vitro with gallic, 2,3,4-trihydroxybenzoic and 3-O-methylgallic acids is fully in agreement with the results previously reported using the intact animal 6. Thus, with the expenditure of a single methyl Biochim. Biophys. Ac/a, 65 (I962) 495 505
500
M . S . MASRI, A. N. BOOTH, F. D E E D s
group, the 2 potential ortho-dihydroxy configurations of gallic acid or 2,3,4-trihydroxybenzoic acid were simultaneously abolished by selective p- or m-O-methylation respectively. In contrast, with 3-0-methylgallic acid, which has but a single orthodihydroxy configuration, either m- or p-O-methylation occurred. With 4-0-methylgallic and 3-methoxy-2,4-dihydroxybenzoic acids, neither of which has an orthodihydroxy configuration, further O-methylation of these substrates was not observed. The a-, fl- and y-isomers of resorcylic acid which lack an ortho-dihydroxy group failed to yield methoxy derivatives in vitro. O-Methylation of the ortho-dihydroxy compound, 2,3-dihydroxybenzoic acid, also failed to occur in vitro, although a slight amount of O-methylation occurred in vivo (unpublished data). The results obtained with the catecholic phenols, pyrogallol and 1-0-methylpyrogallol were dissimilar to those obtained with the catecholic acids : with the phenols conjugation rather than methylation was the evident reaction in vitro with liver slices. The formation of conjugates indicated the liver slices were surviving in the presence of these phenols. These results are consistent with our observation that 50 mg of pyrogallol administered to a rat b y stomach tube resulted in the urinary excretion of large amounts of conjugates, and a trace of 2-0-methylpyrogallol. Similarly, conjugates but no 2,3-dimethoxyphenol were excreted in the urine following administration b y stomach tube of IOO mg of I-0-methylpyrogallol. In contrast ARCHER 6t al. 19 found incubation of pyrogallol with catechol 0-methyl transferase yielded 1-0-methyl pyrogallol and 2,3-dimethoxyphenol. Although pyrogallol is subject to O-methylation in this isolated enzyme system, it does not appear that the same metabolic pathway represents the manner in which the intact organism handles this compound. In this respect, the liver-slice technique, by virtue of its greater degree of integration, suffers less from the rather altificial nature of the technique in vitro. The difference in results obtained by incubation with surviving liver slices, and with isolated O-methyl transferase, as well as differences reported in this paper between the results of the liver-slice technique and those obtained with the intact animal emphasize the hazards of transferring results in vitro to the intact animal. REFERENCES N. BOOTH, C. W. MURRAY, F. D E E D s AND F. T. JONES, Federation Proc., 14 (1955) 32 E. N. BOOTH, C. W T. MURRAY, F. T. JONES AND F. D E E D s , J. Biol. Chem., 223 (1956) 251. D E E D s , A. N. BOOTH AND F. T. JONES, J. Biol. Chem., 225 (1957) 615. N. BOOTH, O. H . EMERSON, F. T. JONES AND V. D E E D s , J. Biol. Chem., 229 (1957) .51. S. MASRI, A. N. BOOTH AND F. D E E D s , Arch. Biochem. Biophys., 85 (1959) 284. N. BOOTH, M. S. MASRI, D. J. ROBBINS, O. H . EMERSON, F. T. JONES AND F. D E E D s , J. Biol. Chem., 234 (1959) 3o14. 7 K. N. F. SHAW, A. McMILLAN AND M. D. ARMSTRONG, J . Biol. Chem., 226 (t957) 255. s M. D. ARMSTRONG, A. McMILLAN AND K . ~N. F. SHAW, Biochim. Biophys. Acta, 25 (1957) 422. 9 j . AXELROD AND R . TOMCHICK, J. Biol. Chem., 233 (1958) 7o2. lO j . PELLERIN AND A. D ' I o R I O , Can. J. Biochem. Physiol., 36 (1958) 491. 11 H . WATANABE, Bull. Agr. Chem. Soc. Japan, 23 (1959) 268. 12 S. SENOH, J. DALY, J. AXELROD AND B. WITKOP, J. Am. Chem. Soc., 81 (19.59) 624o. la j . W . DALY, J. AXELROD AND B. ~VITKOP, J . Biol Chem., 235 (196o) 1155. la M. S. MASRI AND F. D E E D s , Proc. Soc. Exptl. Biol. Med., 99 (1958) 707 . 15 H. A. KREBS AND K. HENSELEIT, I . physiol. Chem. Hoppe-Seyler's, 21o (I932) 33. 1~ A. N. BOOTH, F. T. JONES AND V. D E E D s , J. Biol. Chem., 23o (19.58) 6 6 t . 17 K. N. F. SHAW AND J. TREVARTHEN, Nature, t82 (1958) 79718 S. L. TOMPSETT, J. Pharm. and Pharmacol., I I (1959) 32. 19 S. ARCHER, A. ARNOLD, R. K. KULLNIG AND D. \ ¥ . \¥YLIE, Arch. Biochem. BiophJ's., '~7 (I96O) 153. 1 A. 2 A. 3 F. 4 A. 5 M. 8 A.
Biochim. Biophy,~. ~4cta. (~5 ( 106- J 495 5o5