Species Differences in the Conjugation of 4-Hydroxy-3-Methoxyphenylethanol with Glucuronic Acid and Sulphuric Acid

Biochem. J. (1976) 158, 33-37 33 Printed in Great Britain

Species Differences in the Conjugation of 4-Hydroxy-3-methoxyphenylethanol with Glucuronic Acid and Sulphuric Acid By KIM PING WONG Department ofBiochemistry, University ofSingapore, Singapore 3 (Received 24 September 1975) The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions ofliver respectively. These two conjugates were also produced simultaneously byusingthe post-mitochondrial fraction ofrat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; therate ofsulphation of4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supematant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxyphenylglycol were also formed by enzyme preparations ofrabbit adrenal andrat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22mM and that for Na2SO4 was 3.45mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenylethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3mM. It was concluded that sulphate conjugation of 4-hydroxy-3- methoxyphenylethanol predominates in most species of animals.

4-Hydroxy-3-methoxyphenylethanol is a meta- methoxyphenylglycol (piperazine salt, 99 % pure) bolite of dopa (3,4-dihydroxyphenylalanine) and were purchased from Aldrich Chemical Co., dopamine (3,4-dihydroxyphenethylamine) (Goldstein Milwaukee, WI, U.S.A. UDP-glucuronic acid et al., 1960). After administration of dopa or (triammonium salt, 99% pure), ATP (sodium salt, dopamine, 4-hydroxy-3-methoxyphenylethanol was 99 % pure), D-glucuronic acid, D-glucuronolactone, found in the blood and central nervous system fl-glucuronidase (type B-1, prepared from bovine (Goldstein & Gerber, 1963), where inactivation was liver, containing 500000 Fishman units per g ofsolid) thought to occur by conjugation (Goldstein, 1964). were obtained from Sigma Chemical Co., St. Louis, It is present in normal human urine (Goldstein et al., MO, U.S.A. UDP_[U_'4C]glucuronic acid (specific 1961) and may be quantitatively determined by g.l.c. radioactivity 290 or 302mCi/mmol) and Na235SO4 (Karoum et al., 1971; Braestrup, 1972). From these (specific radioactivity 24 or 67mCi/mmol) were determinations, which include hydrolysis, it was purchased from The Radiochemical Centre, Amer- inferred that the alcohol occurs in brain and urine as a sham, Bucks, U.K. conjugate, the nature of which is unknown. Studies with sulphatase and fl-glucuronidase have shown that Methods a sulphate conjugate of the alcohol predominates in human urine, whereas the ratexcreted this compound Preparation of enzymes mainly as a glucuronide (Karoum et al., 1973). The Adult male animals were used. The rats were ofthe sulphate conjugate was identified in rat brain after albino Wistar strain, and the other animals were administration of Na235SO4 (Eccleston & Ritchie, local hybrids. Their body weights were as follows: 1973). The present study was undertaken to investi- rat, 150-200g; rabbit, 1-1.2kg; mouse, 25-30g; gate the conjugation of 4-hydroxy-3-methoxyphenyl- guinea pig, 200-300g. A liver biopsy (150mg) was ethanol in vitro in different tissues and in different obtained from a male patient (T.C.S.) aged 62, animal species. suffering from moderately differentiated adeno- carcinoma of the descending colon. The biopsy was Materials normal in appearance. (a) Glucuronyltransferase. This enzyme was pre- 4 - Hydroxy - 3 - methoxyphenylethanol (homo- pared by the procedure of Wong & Sourkes (1967). vanillyl alcohol, 99 % pure) and 4-hydroxy-3- The microsomal pellet obtained after centrifugation Vol. 158 2 K. P. WONG of the post-mitochondrial fraction at 105OOOg for 1 h were counted for radioactivity in 5ml of scintillator was suspended in ice-cofd 0.15M-KCI so that 1 ml of containing 0.4% 2,5-diphenyloxazole and 0.025% this suspension corresponded to I g fresh weight of 1,4-bis-(5-phenyloxazol-2-yl)benzene in toluene. tissue. This was subjected to overnight dialysis at 4°C The radioactivity of 4-hydroxy-3-methoxyphenyl- against lOmM-EDTA (disodium). ethanol [14C]glucuronide or 4-hydroxy-3-methoxy- (b) Sulphate-activating and sulpho-transferring phenylethanol [35S]sulphate or both were measured. enzymes. The supernatant obtained above is referred Standards of Na235SO4 and UDP-[U-14C]glucuronic to as the high-speed supernatant; it contains these acid were chromatographed and similarly counted enzymes. for radioactivity. (c) Post-mitochondrial fraction. A 10 % (w/v) homogenate of liver, kidney, adrenal or brain was Hydrolysis of biosynthetic 4-hydroxy-3-methoxy- prepared in cold 0.15M-KCI. Centrifugation of this phenylethanol glucuronide homogenate at 150OOg for 30min removed the The material contained in the radioactive peak mitochondria leaving the supernatant as the post- thought to be 4-hydroxy-3-methoxyphenylethanol mitochondrial fraction. glucuronide was eluted from ten paper strips, each with radioactivity greater than 10000 c.p.m. The RF Reaction conditions value of this peak was not determined, as the solvent (a) Glucuronidation. The incubation medium was front had moved off the chromatogram. However, the same as decribed previously (Wong, 1971), this conjugate was characterized as a peak at 19.5cm except for the aglycone, which was 4-hydroxy-3- from the origin on the chromatogram developed by methoxyphenylethanol in this case. The medium the descendimg technique at room temperature (290C) contained, in a final volume of 200,ul, the following in solvent A for 26h. One portion ofthe above eluate (final concentrations in parentheses): 4-hydroxy-3- was incubated overnight at pH 5.0 with bovine liver methoxyphenylethanol (2mM); MgCI2 (5mM); UDP- ,8-glucuronidase (5000 Fishman units). Another was [U-14C]glucuronic acid (3.32 or 6.64pm) and/or un- boiled at 100°C for 1 h with 6M-HCI and the third was labelled UDP-glucuronic acid (1mm) and 0.5M- untreated. Then 50Ol of each was chromatographed Tris/HCl buffer, pH7.8. The reaction was started on paper and developed in solvent B. Standards of with 50,ul of the rabbit liver microsomal preparation, authentic 4-hydroxy-3-methoxyphenylethanol, D- containing 3.7mg of protein/ml. The protein content glucuronicacidandD-glucuronolactonewerechroma- was detemined by the procedure of Lowry et al. tographed simultaneously. The free alcohol was (1951). located with Folin-Ciocalteu reagent (Waldi, 1965) (b) Sulphation. The procedure described for the or diazotized sulphanilic acid (Smith, 1960), and sulphation of 4-hydroxy-3-methoxyphenylglycol glucuronic acid and its lactone were detected with (Wong, 1975) was followed, but 4-hydroxy-3- naphtharesorcinol reagent (Smith, 1960). To identify methoxyphenylethanol [35S]sulphate was measured. the alcohol released from biosynthetic 4-hydroxy-3- (c) Simultaneous glucuronidation and sulphation. methoxyphenylethanol glucuronide, a large amount For this, SOl of the post-mitochondrial fraction of of the unlabelled conjugate was first produced and liver, kidney or brain was used. The incubation subjectedtoacidandenzymichydrolysis.Thehydroly- medium contained 4-hydroxy-3-methoxyphenyl- sates were chromatographed on cellulose-coated ethanol (2mm), MgCl2 (8mM) dissolved in dithio- t.l.c. plates in solvent B. threitol (3mM), 0.5M-Tris/HCI buffer (pH8.0), UDP-[U-14C]glucuronic acid (3.32 or 4.32,uM), Results ATP (8 mM) and Na235SO4 (0.65 or 3.1 mM). For all the three systems above, incubation was Formation and hydrolysis of 4-hydroxy-3-methoxy- carried out at 37°C in a metabolic shaker, the times of phenylethanol glucuronide incubation being 10 or 15min for glucuronidation, 4-Hydroxy-3-methoxyphenylethanol [14C]glucuro- 30min for sulphation and 15 or 30min for simultane- nide was synthesized in vitro by the transfer of ous glucuronide and sulphate conjugations. The 114C]glucuronic acid from UDP-[U-14C]glucuronic reactions were stopped by adding 501 each of ad to the alcohol. Under the same experimental ZnSO4 (10%, w/v) and Ba(OH)2 (0.3M). The precipi- conditions, 4-hydroxy-3-methoxyphenylglycol was tate was removed by centrifugation, and 25 or SOpl also glucuronidated. The chromatographic procedure of the supernatant was chromatographed on What- used for the separation of the glucuronide was used man no. 1 paper (56cmx 1.2cm) by the descendi but the time of development was extended to 40h technique in solvent A [propan-2-ol/NH3/water The distance traversed by this glucuronide and (8:1 :1, by vol.)] for preparations (a) and (c) and in UDP-glucuronic acid were 16.5 and 3.5cm respec- solvent B [butan-l-ol/acetic acid/water (4:1 :5, by tively from the origin. vol., upper phase)] for preparation (b). After the Treatment of unlabelled 4-hydroxy-3-methoxy- chromatogram had been developed, 2cm fractions phenylethanol glucuronide with Ii-glucuronidase 1976 GLUCtURONIDATION AND SULPHATION OF HOMOVANILLYL ALCOHOL 35 liberated 4-hydroxy-3-methoxyphenylethanol, which hydrolysis studies carried out- on 4-hydroxy-3- gave a purple coloration with the Folin reagent and methoxyphenylglycol sulphate (Wong, 1975) were orange coloration with diazotized sulphanilic acid. performed on labelled and unlabelled 4-hydroxy-3- This aglycone has RF 0.89 on a cellulose-coated t.l.c. methoxyphenylethanol sulphate, Na235SO4 and 4- plate developed in solvent B. Labelled glucuronic hydroxy-3-methoxyphenylethanol were shown to be acid released from 4-hydroxy-3-methoxyphenyl- products of acid hydrolysis, indicating that the ethanol [(4C]glucuronide after acid and fl-glucuroni- conjugate formed was 4-hydroxy-3-methoxyphenyl- dase hydrolysis was located as a radioactive peak ethanol sulphate. with RF 0.18 on a paper chromatogram developed in solvent B. This peak was coincident with that of Kinetic data on the sulphation of 4-hydroxy-3- authentic glucuronic acid. In the acid hydrolysate, methoxyphenylethanol by rabbit liver another radioactive peak with RF 0.41 was observed. Measurement ofKm of4-hydroxy-3-methoxyphenyl- This presumably contained glucuronolactone, which ethanolfor the sulphotransferase reaction. The 'active' hasthe same mobility in this solvent system. Conceiv- sulphate adenosine 3'-phosphate 5'-sulphatophos- ably, lactonization had occurred as a result of boiling phate was first generated from Na23SO4 by using the with HCI. high-speed supernatant of rabbit liver (Wong, 1975). The reaction was stopped by boiling. Before the Kinetic data on the transglucuronidation of4-hydroxy- second incubation in which 35SO4 from adenosine 3-methoxyphenylethanol by rabbit liver microsomal 3'-phosphate 5'-[35S]sulphatophosphate was trans- fractions ferred to 4-hydroxy-3-methoxyphenylethanol, Effect of pH. The pH optimum for the trans- another portion of the same enzyme preparation was glucuronidation of 4-hydroxy-3-methoxyphenyl- added with introduction of EDTA to inhibit the ethanol was 8.2, when 0.5M-Tris/HCI buffer was sulphate-activating system (Brunngraber, 1958). used. The Km for 4-hydroxy-3-methoxyphenylethanol Effect of time of incubation. The formation of obtained from the double-reciprocal plot was 4-hydroxy-3-methoxyphenylethanol [14C]glucuron- 0.036mM. ide increased with-time ofincubation. Linear velocity Measurement of Km of Na2SO4. Adenosine 3'- was obtained for reaction up to 15min. phosphate 5'-[35S]sulphatophosphate formed from Effect ofenzyme concentration. There was increased Na235SO4 by the high-speed supernatant preparation formation of 4-bydroxy-3-methoxyphenylethanol of rabbit liver was measured by the transfer of its [14Clglucuronide with increasing concentration of sulphate to 4-hydroxy-3-methoxyphenylethanol and enzyme. harmol (Wong, 1974). With both substrates, the Km Measurement ofK. of4-hydroxy-3-methoxyphenyl- value for Na2SO4 was 3.45 mM. ethanol. The rate of reaction over the concentration range 0.02-2mi was determined. In one experiment, Sulpho-conjugation of 4-hydroxy-3-methoxyphenyl- only radioactive UDP-glucuronic acid at a final ethanol by kidney, brain and adrenal concentration of 6.644p was used and in the other This was measured in the high-speed supernatant 1 mM unlabelled nucleotide was also added. The Km and 10% homogenate preparations; the rates of values for 4-hydroxy-3-methoxyphenylethanol ob- sulphation by mouse kidney were 1.8 and 1.84 times tained from Lineweaver-Burk (1934) plots of the greater than the rates with liver preparations. From above sets ofdata were 1.33 and 1.25mM respectively. this, it was inferred that the sulpho-conjugating Measurement ofKm ofUDP-glucuronic acid. At very activity measured in the 10% homogenate reflects low nucleotide concentration (0.42-16.6,pM), a linear closely its activity in the high-speed supernatant relationship was established between the rate of for- preparation. Neither the rat nor rabbit kidney mation of 4-hydroxy-3-methoxyphenylethanol glu- formed4-hydroxy-3-methoxyphenylethanol sulphate. curonide and the nucleotide concentration up to Of the other tissues examined, rat, but not mouse 3.3AuM. The addition of unlabelled UDP-glucuronic brain, and rabbit adrenal were able to conjugate acid (0.01-1 mM) at a fixed concentration (3.32pM) of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy- radioactive UDP-glucuronic acid increased the trans- 3-methoxyphenylglycol with sulphate, but the small glucuronidation reaction progressively. A Line- and large intestines of rat did not show any activity. weaver-Burk plot of the second set of data gave a The amount of glycol sulphate formed by rat brain Km of 0.22mM for UDP-glucuronic acid. was 4.73 times more than 4-hydroxy-3-methoxyphenylethanol sulphate. Formation and hydrolysis of 4-hydroxy-3-methoxyphenylethanol sulphate Simultaneous formation of 4-hydroxy-3-methoxy- The sulphate conjugate has RF 0.48 on a paper phenylethanolglucuronide and sulphate chromatogram developed in solvent B. When the The sulphation and glucuronidation was further Vol. 158 36 K. P. WONG confirmed in a single reaction with crude 10% homo- Discussion genate or post-mitochondrial fraction of liver of rat, rabbit or guinea pig. Of all the tissues tested, rat liver There is a distinct pattern of conjugation of was the most suitable for this simultaneous reaction 4-hydroxy-3-methoxyphenylethanol in different ani- as it contained relatively high activity of both mals. The liver was the only organ capable offorming transferases. The chromatographic profile of Fig. 1 the glucuronide, but, in addition to liver, kidney, showed the complete separation of the glucuronide brain and adrenal ofvarious animals could synthesize and sulphate peaks from their labelled precursors. It the sulphate conjugate. The high affinity of sulpho- must be emphasized that the reaction conditions used transferase for the glycol accounted for its presence here are not optimum for both reactions. Thus the exclusively as the sulphate conjugate in rat brain conjugates formed simultaneously were not deter- (Schanberg et al., 1968). Mouse brain on the contrary mined quantitatively. This system is, however, is devoid ofthis activity, which explained the absence extremely suitable for studying the overall pattern of of the glycol sulphate from its cerebral tissue conjugation of 4-hydroxy-3-methoxyphenylethanol. (Sharman, 1973). Whether the high renal activity in Mouse and cat liver did notform any glucuronide, but mouse facilitates the clearance of the glycol and the sulphate conjugate was produced in appreciable alcohol remains to be studied. amounts. In contrast, a 10% homogenate of human In general, most animals seem to form the sulphate liver showed formation of 4-hydroxy-3-methoxy- conjugate preferentially as far as the catecholamines phenylethanol glucuronide, but the sulphate con- and their metabolites are concerned. Measurements of jugate was not produced. Thus the modes of conju- the urinary concentrations of these compounds and gation of 4-hydroxy-3-methoxyphenylethanol by studies in vivo tend to substantiate this generalization, hepatic tissues differ in various animals. e.g. the sulpho-conjugates of the following have been reported: adrenaline (Richter, 1940), dopamine (Jenner & Rose, 1973), 4-hydroxy-3-methoxyphenylglycol (Schanbergetal., 1968), 4-hydroxy-3-methoxyphenylethanol (Goldstein et al., 1960; Eccleston & Ritchie, 1973), dihydroxyphenylglycol (Eccleston & 400 t) ITvv r Ritchie, 1973) and 3-0-methyl-adrenaline and -nor- 'a i adrenaline (Sharman, 1973). An analysis of the 0 A sulphate and glucuronide values in these references _3 t showed that in most cases the former predominates. 300 In this study, rabbit hepatic sulphotransferase has 36 times greater affinity for 4-hydroxy-3-methoxyphenylethanol than does glucuronyltransferase. Thus co ::: when the concentration of this substrate is limiting, : v 200 >. * sulphation would undoubtedly be the preferential r- route ofconjugation ofthis alcohol. With harmol, an a 0 exogenous substrate, the sulphate conjugate was also cd c formed more in vivo & 0 x readily (Mulder Hagedoorn, u" 100 1974) and in vitro (Mulder, 1975). en The significance of the sulpho-conjugates of z 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy- x 3-methoxyphenylglycol in brain metabolism deserves 1- o 0 further investigation. The metabolic sequence pro- ao o0 20 30 40 posed for these neutral metabolites of the catechol- Distance traversed in 26h (cm) amines, i.e. their passage out of the brain with sub- sequent peripheral conjugation and final return to the Fig. 1. Radiochromatogram showing the biosyntheses of brain (Taylor & Laverty, 1969), may occur in the 4-hydroxy-3-methoxyphenyethanoI 14C]glucuronide (peak but it is of in B) and 4-hydroxy-3-methoxyphenylethanol [35S]sulphate mouse, probably secondary importance (peak C) by rat liver the rat whose cerebral tissue possesses the entire sulpho-conjugatory machinery (Wong, 1975). This These peaks are separated from the labelled precursors alcohol is believed to be of low significance in brain UDP-[U-_4C]glucuronic acid and Na235SO4 (peak A), metabolism (Braestrup, 1973) and it is present in very both of which have the same mobility in solvent system A. low concentration in brain and cerebral-spinal fluid The ordinates show the radioactivity (c.p.m.) per fraction of the chromatogram: left ordinate ( ) and right (Karoum et al. 1971; Braestrup, 1972). As peripheral ordinate (....). The abscissa shows the distance traversed in tissues could conjugate this alcohol with glucuronic 26h by radioactive compounds during descending chroma- acid and/or sulphuric acid, it would be interesting to tography. find out if these conjugates participate as active 1976 GLUCURONIDATION AND SULPHATION OF HOMOVANILLYL ALCOHOL 37 metabolic intermediates, both centrally and peri- Jenner, W. N. & Rose, F. A. (1973) Biochem. J. 135, 109- pherally. In particular, the fates of the sulphates of 114 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy- Karoum, F., Ruthven, C. R. J. & Sandler, M. (1971) in should Biochem. Med. 5, 505-514 3-methoxyphenylglycol brain metabolism Karoum, F., LeFRvre, H., Bigelow, L. B. & Costa, E. be examined since the latter was found in rat brain (1973) Clin. Chim. Acta 43, 127-137 exclusively in this form. Lineweaver, H. & Burk, D. (1934) J. Am. Chem. Soc. 56, 658-666 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, I thank Miss Theresa Yeo Huay Cheng for her skilful R. J. (1951) J. Biol. Chem. 193, 265-275 and diligent technical assistance and the Wellcome Trust Mulder, G. J. (1975) Anal. Biochem. 64, 350-360 for financial support. I also thank Mr. Ho Soon Teck of Mulder, G. J. & Hagedoom, A. H. (1974) Biochem. the Department of Surgery, University of Singapore, for Pharmacol. 23, 2101-2109 the specimen of human liver. Richter, D. (1940) J. Physiol. (London) 98, 361-374 Schanberg, S. M.,Schildkraut,J. J., Breese, G. R. &Kopin, I. J. (1968) Biochem. Pharmacol. 17, 247-254 Sharman, D. F. (1973) Br. Med. Bull. 29, 110-115 References Smith, I. (ed.) (1960) Chromatographic and Electro- Braestrup, C. (1972) Biochem. Pharmacol. 21, 1775-1776 phoretic Techniques, vol. 1, pp. 291-307, Interscience, Braestrup, C. (1973) J. Neurochem. 20, 519-527 New York Briinngraber, E. G. (1958) J. Biol. Chem. 233, 472-477 Taylor, K. M. & Laverty, R. (1969) J. Neurochem. 16, Eccieston, D. & Ritchie, I. M. 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