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-methoxy- phenylethanol was demonstrated in vitro by using the high-speed supernatant and micro- somal fractions ofliver respectively. These two conjugates were also produced simultane- ously 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-methoxy- phenylglycol 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-methoxyphenyl- ethanol 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.