UDP-glucuronyltransferase activity toward harmol in human liver and human fetal liver cells in culture

ANALYTICALBIOCHEMISTRY

185,44-50

(1990)

UDP-Glucuronyltransferase Activity toward Harmol in Human Liver and Human Fetal Liver Cells in Culture Theresa

M. C. Tan, K. H. Sit,* and Kim

Ping

Wang’

Departments of Biochemistry and *Anatomy, National University of Singapore, lo-Kent Ridge-Crescent, Singapore 0511, Singapore

Received

June

20,1989

MATERIALS

This paper presents a fast HPLC assay for measuring UDP-glucuronyltransferase (UDPGT) activity in extracts of adult human liver and human fetal liver cells in culture. Harm01 glucuronide formed was quantitated directly without prior hydrolysis after a simple step of selective extraction of harmol. The method is applicable to crude liver homogenates as well as to partially fractionated preparations. It is several fold more sensitive than the conventional detection of harm01 glucuronide by TLC, making it possible to distinguish the low and high affinity forms of UDPGT of adult human liver and to detect the low affinity form in fetal cells. The possible participation of both forms of GT in adults under different conditions and the apparent lack of the high affinity form in the fetal liver is discussed. Q ISSO Academic Press,

Inc.

Harm01 (7-hydroxy-l-methyl-SH-pyrido-[3,4b]indol) has been used extensively for studies of sulfate and glucuronide conjugation in the rat as it is subjected to only these phase II biotransformations (l-5). Routinely, TLC has been used to separate harm01 from its conjugates (1,6) and radiolabeled harm01 has also been employed to enhance the sensitivity of the method (7). Analysis of harm01 and its conjugated metabolites by HPLC required enzymatic hydrolysis before the conjugates could be measured (8). In this paper, a direct method for the quantification of harm01 glucuronide by HPLC with fluorometry is presented to study the kinetics of the glucuronidation reaction in the microsomal fraction of human liver and in extracts of human fetal liver cells in culture.

’ To whom

44

correspondence

should

be addressed.

AND

METHODS

Materials. Harm01 hydrochloride, uridine diphosphoglucuronic acid, ammonium salt, &glucuronidase (Helix pomatia, type H- 1) , and sodium octyl sulfate were purchased from Sigma Chemical Co., methanol (HPLC grade) from J. T. Baker, and TLC plates precoated with cellulose from Merck. Hepatic liver samples. In this study, two samples of human liver were used. The first was collected on ice 10 h postmortem and has been kept at -80°C for the past 4 years. The second was a relatively fresh biopsy sample. A 20% (w/v) homogenate of liver was prepared in 0.15 M KC1 containing 3 mM DTT2 and centrifuged at 15,000g for 30 min and 108,OOOgfor 1 h to provide the respective high-speed supernatant and cytosolic fractions. The final pellet which represented the microsomal fraction was dialyzed overnight at 4°C in 10 mM EDTA which had been adjusted to pH 8.2, according to the procedure reported previously (9). All these different enzyme fractions were stored in small aliquots of 100 to 500 ~1 at -80°C. Extracts from human fetal liver cells. Livers from second trimester prostaglandin-induced abortuses were used. The conditions of culture were as described (10). A 50% (w/v) suspension of cells in 0.15 M KC1 containing 3 mM DTT was prepared and stored at -80°C in small aliquots of 0.1 ml. Assay of UDPGT activity in adult human liver. A typical reaction mixture contained the following with final concentrations in parentheses: 10 ~1of dialyzed microsomal fraction (containing 25 pg protein), 2.5 ~1 of 100 mM Mg2+ (5 mM), 5 ~1of 10 mM UDPGA (1 mM), 2.5 ~1of 5 mM harm01 (250 PM), and 30 ~1 of 50 mM glycine2 Abbreviations used: UDPGA, UDPGT, UDP-glucuronyltransferase; DTT, dithiothreitol; RFU, relative

uridine

diphosphoglucuronic GT, glucuronyltransferase; fluorescence units.

acid;

0003~2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

CHROMATOGRAPHIC

ASSAY

OF

NaOH buffer, pH 8.6, to make up a final volume of 50 ~1. The reaction was allowed to proceed at 37°C for 20 min. It was terminated by the addition of 10 ~10.3 M Ba(OH)2 and 10 ~1 of 5% ZnSOl. Controls in which UDPGA was omitted were similarly incubated. After centrifugation, 10 ~1 of the supernatant was spotted for TLC analysis. For quantification by HPLC, the supernatant was diluted 20 times with distilled water, centrifuged, and filtered through a Millex filter (Millipore) of 0.45-pm pore size and 10 ~1 was injected for analysis. Assay of UDPGT activity in extracts of human fetal liver cells. Conditions essentially similar to those above were used except that an additional extraction step was introduced to remove selectively the substantial amount of unreacted harm01 which remained in the incubate. This was due to the lower UDPGT activity in fetal cell extracts compared to the microsomal preparation of adult liver. A higher concentration of UDPGA of 5 mM was also employed and the reaction was started with 15 ~1 of the cell extract which contained 150-200 pugprotein in a final volume of 100 ~1. The reaction was terminated with 0.3 M Ba(OH)2 and 5% ZnSO,, and the incubate was adjusted to 250 ~1 with water. To this was added 150 ~1 of 0.2 M sodium phosphate buffer, pH 9.5. It was extracted twice with 0.5 ml of ethyl acetate following the procedure of Pang et al. (7). The aqueous layer was centrifuged and filtered and 20 ~1 was injected for quantification of harm01 glucuronide by fluorometry. Thin-layer chromatography and high-performance liqHaruid chromatography with fluorometric analysis. mol was separated from harm01 glucuronide by TLC according to the method described (6). The fluorescence of the eluates was read in a Perkin-Elmer luminescence spectrometer (Model LS-5) at excitation and emission wavelengths of 310 and 420 nm, respectively. This procedure was not sensitive for the measurement of UDPGT activity in the fetal liver cells in culture. The constant flow HPLC (Waters) consisted of a Model 1000A solvent delivery system, a Model U6K injector, a Whatman CSKI guard column packed with Pellicular ODS (37- to 53-pm particle size) and a microbore column (100 X 2.1 mm) containing 5 Frn Hypersil ODS. The mobile phase contained 50% methanol and 50% 20 mM KH,PO, with 2.5 mM sodium octylsulfate at pH 3.5. The flow was maintained at 0.3 ml/min with a back pressure registered between 1500 and 2000 psi. Detection was by means of a programmable fluorescence detector (Hewlett-Packard Model 1046) which was in turn connected to an HP 3390 integrator. Normal settings for the fluorescence detector were: gain = 8 or 9 with response time setting of 4. All injections were done in duplicate or occasionally in triplicate. Various volumes of 5 to 25 ~1 of a 1 pM harm01 solution (corresponding to 5 to 25 pmol) were injected under sim-

UDP-GLUCURONYLTRANSFERASE

45

ilar conditions for standardization. The HP 3390 integrater was used with area mode operation. Isolution and identification of harm01 glucurode. Harm01 glucuronide was synthesized from UDPGA and harmol, using a microsomal fraction of rat liver prepared in a manner similar to that described above. The glucuronide conjugate was separated from harm01 by passing the reacted incubate through a column packed with Sephadex G-25 as described previously (11). The eluate containing harm01 glucuronide was subjected to hydrolysis by 20 units of /3-glucuronidase at pH 5.0. A control with the omission of @-glucuronidase was included for comparison. Both unreacted harm01 glucuronide and harm01 liberated in the hydrolysate were identified by HPLC-fluorometry. Determination of the relative molar fluorescence of harmol to harm01 glucuronide. This was necessary as harmol glucuronide is not available commercially. The standard GT assay procedure was employed using 50 pM harmol. This suboptimal concentration of acceptor was chosen so that the quantitation of both harm01 and its glucuronide could be carried out for each analysis without changing the sensitivity setting, i.e., the gain value on the HP 1046 fluorescence detector. At timed intervals from 0 to 50 min, the reaction was terminated. The incubated mixtures were subjected to HPLC analyses. This ratio was determined by finding the gradient from the plot of the area of the harm01 peak against the area of the glucuronide peak. Each plot had eight points, derived by the time curve. Using the HPLC method, the average ratio determined in five sets of experiments was 0.670 +- 0.033 and the correlation within each set was at least 90% (ranging from 89.9 to 99.3%). Given this correlation factor, the amount of harm01 glucuronide formed was extrapolated from a standard of harmol. UDPGT activity in various fractions. Ten microliters of each of the following fractions from human liver was used, with the protein content (given in parentheses) determined by the Lowry procedure (12): crude homogenate (25 pg), 15,OOOg supernatant (155 pg), the cytosolic or 108,OOOg supernatant (150 pg), and the dialyzed microsomal fraction (25 pg) . RESULTS Identification of harm01 glucuronide by HPLCfluorometry. Typical HPLC chromatograms are shown in Fig. 1. The retention times for harm01 glucuronide and harm01 were, respectively, 1.7 and 4.0 min for the reacted incubates which were injected directly (Fig. lb). Extraction with ethyl acetate produced a diminished harm01 peak without affecting the peak height or fluorescence of harm01 glucuronide. Each HPLC analysis could thus be accomplished in 8 min, by which time the baseline was reestablished. In controls where UDPGA

46

TAN,

a

SIT,

b H

HG

c

H

II 8min

C

d H( 5

H b FIG. 1. HPLC of (a) the control in which (b) a typical reaction incubate, showing the uronide (HG) from harm01 (H). Harm01 was the above respective assay incubates with (c)and(d).

UDPGA was omitted and separation of harm01 glucselectively extracted from ethyl acetate as shown in

was omitted, there was no formation of harm01 glucuronide (Figs. la and lc). By stop-flow analysis, the excitation and emission spectra of both harm01 and harm01 glucuronide present in the reacted mixture were examined. As their excitation and fluorescence spectra are almost identical (Fig. 2), all HPLC analyses and quantitation were monitored at an excitation wavelength of 310 nm and an emission wavelength of 420 nm. The identity of harm01 glucuronide was confirmed by comparison with the spectra and the retention time of pure harm01 glucuronide isolated by gel-filtration with Sephadex G-25, and after it had been subjected to the action of fl-glucuronidase which liberated harmol. Based on a solution of known concentration of harm01 glucuronide, quantified by HPLC as described under

AND

WONG

Materials and Methods, the sensitivities of the HPLC and TLC methods were compared. The minimum amount of harm01 glucuronide detectable by HPLC was 2 pmol, while at least 25 pmol was required for detection by TLC with confidence. All reacted incubates were subjected to HPLC analysis unless otherwise stated. HPLC was the method of choice as the TLC procedure was less sensitive and controls in which UDPGA was omitted yielded some basal fluorescence readings (corresponding to RFU of 1 to 4) making the detection of low concentrations of harm01 glucuronide unreliable. Harm01 glucuronide could not be detected with the fetal liver cells using the TLC procedure. Kinetic studies of human liver UDPGT. The pH activity was investigated using 0.05 M KH2P04-NaOH buffer (pH 6.6-8.0) and 0.05 M glycine-NaOH buffer (pH 8.4-9.1). The pH optimum observed was 8.6 and the rate of reaction was linear for 20 min and for microsomal protein up to 25 pg/assay. The effects of 2 to 10 mM Mg2+ on UDPGT activity were examined. Maximal activation was observed from 5 to 10 mM and at these concentrations, the GT activity was 2.5 times higher than that of controls where Mg was omitted from the incubate. Analysis of kinetic data for harm01 from 0.5 to 250 PM and UDPGA between 10 PM and 1 mM using the EadieHofstee plots (Figs. 3a and 3b) generated by the Enzpack software (13) showed that two affinity sites may be involved for each of these substrates (Table 1). High concentrations of harm01 and UDPGA were found to be inhibitory, with 16% inhibition at 375 PM harm01 and 35% at 10 mM UDPGA. At the lower concentration range of both substrates, the glucuronide formed could not be detected by TLC. The apparent K, values for harm01 and UDPGA, measured at higher concentrations of these substrates (see Figs. 4a and 4b) seemed to correlate more closely with the values for the low affinity form established by the HPLC procedure (Table 1). Specific activities of human liver extracts. The specific activities of various fractions of human liver were determined and are shown in Table 2. There was low activity in both the 15,000g and the 108,OOOg supernatant fractions. The activity in the crude homogenate was only slightly lower than that in the microsomal fractions. It is interesting to note that the GT activity was retained after storage at -80°C for 4 years. UDPGT activity in extracts of human fetal liver cells in culture. The pH activity was investigated using the same buffers as above. The optimum pH was also 8.6 and the rate of reaction was linear for 20 min and with cellular protein up to 200 fig/assay. Analysis of the kinetic data by the Eadie-Hofstee plots for harm01 from 10 to 250 /IM and UDPGA from 200 PM to 5 mM revealed

CHROMATOGRAPHIC

ASSAY

OF

UDP-GLUCURONYLTRANSFERASE 18

a

b 16

6

0-l

260

I

I

I

I

I

,

280

300

320

340

360

380

excitation

7

I

I

I

/

I

I

I

I

I

emission

(b) spectra of harm01 (m . . .m) and harm01 310 and 420 nm, respectively.

glucuronide

(0 -

wavelength 0) as recorded

by stop-flow

HPLC

are

only the low affinity form (Figs. 5a and 5b and Table 1). No harm01 glucuronide could be detected at the low concentration range of harm01 (from 0.5 to 5 PM), the concentration range required for establishing the high affinity form. There was inhibition of the UDPGT reaction at high concentrations of harm01 (5 and 16% inhibi-

5

1

340 360 380 400 420 440 460 480 500 520 540

wavelength

FIG. 2. The excitation (a) and emission analysis. Excitation and emission maxima

3

0 I

tion at 375 PM and 1 mM harmol, respectively) and UDPGA (10, 27, and 48% at 10, 15, and 20 mM UDPGA, respectively) as was observed with UDPGT of adult liver. The UDPGT activities at the different stages of culture were also examined. The activity progressively de-

9 11 13 15 17 19 21 23 25 27 (v/s)/pM -’

FIG. 3. Eadie-Hofstee plots of the formation of harm01 glucuronide as determined by HPLC-fluorometry, harm01 (0) and 10 to 250 pM harm01 (0) measured at 1 mM UDPGA, and (b) 10 to 125 pM UDPGA (0) at 100 pM harmol. Velocity, u, was expressed in pmol harm01 glucuronide/min/mg protein.

in the presence of (a) 0.5 to 5 pM and 0.1 to 1 mM UDPGA (0) measured

48

TAN, TABLE

Kinetic

1

Data of UDPGT Activity of Adult Human and Human Fetal Liver Cells in Culture

Parameter measured

SIT, AND WONG

Adult liver

pH optimum Apparent K,,, (j&M) for harm01 (i) High affinity (ii) Low affinity Apparent V,,, (pmol harm01 glucuronide/min/mg protein) (i) High affinity (ii) Low affinity Apparent K, (wM) for UDPGA (i) High affinity (ii) Low affinity Apparent V,,. (pmol harm01 glucuronide/min/mg protein) (i) High affinity (ii) Low affinity

Liver

Fetal liver cells

8.6

8.6

3.15 38.3 (42.4)’

87.6 543

158 473 (394)O

TABLE 2 Specific Activities of UDPGT (Expressed in pmol harm01 glucuronide/min/mgprotein) in Different Fractions of Human Liver and Extracts of Human Fetal Liver Cells in Culture”

51.4

(A) Fractions prepared from adult human liver stored at -8O’C Liver homogenate Microsomal fraction 15,000g Supernatant 108,OOCgCytosolic fraction (B) Microsomal fraction of liver biopsy sample (C) Extracts of quiescent human fetal liver cells (mean k SD of four batches of culture)

966.1 1069.7 242.7 20.2

530.9 34.7 + 2.3

-

19.8

a All enzyme preparations

were frozen and thawed once only.

396

374

-

794

15.4

’ Values determined by TLC analysis.

clined with increasing length of culture and by the 12th day of culture only 59% of the activity remained (Fig. 6). DISCUSSION

Recent studies have shown that different forms of UDPGT exist in the human liver for both phenolic and

carboxylic acceptors (14-17). The presence of high and low affinity forms of GT had been demonstrated with lnaphthol (14) and morphine (16); both substrates were used in their radiolabeled forms for the requisite sensitivity. The two affinity sites were also observed in our study with not only harm01 but UDPGA using the more sensitive HPLC-fluorometric assay (Table 1). Likewise, glucuronidation of harm01 by human fetal liver cells could only be detected by the HPLC-fluorometric procedure, but not by the TLC method; the latter observation was in agreement with the report of Steiner et al. (18). The apparent K, value for harm01 measured in the fetal cells appeared to correspond more closely to the high K, component of the adult liver system. These data concurred with the kinetics of morphine glucuronidation where the fetal liver seemed to have only the low affinity

22

20-

a

20

18-

18 1616 l414 12-

r2 = 0.94 >

12 10

8-

8

6-

6

4-

4

2-

2

0 7

0 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

(V/s)X102/jdul“ FIG. 4. Eadie-Hofstee plots of the formation of harm01 glucuronide analysed by TLC in the presence of (a) 10 to 250 pM harm01 at 1 mM UDPGA and (b) 0.1 to 1 mM UDPGA at 109 pM harmol. Velocity, v, was expressed in arbitrary relative fluorescence units of harm01 glucuronide formed/20 min.

CHROMATOGRAPHIC

ASSAY

OF

49

UDP-GLUCURONYLTRANSFERASE

a

0

0.5

1

1.5

2

2.5

3

3.5

(v/s) x 10’ / j&l -’ FIG. 5. of harm01 protein.

Formation of harm01 glucuronide by extracts of human fetal cells in culture as analyzed by HPLC in the presence of (a) 10 to 250 at 5 mM UDPGA and (b) 200 pM to 5 mM UDPGA at 250 pM harmol. Velocity, u, was expressed in pmol harm01 glucuronide/min/mg

form (19), suggesting that the high affinity form may be absent in the fetal cells and was perhaps developed later. Caution must be exercised in this interpretation which requires further characterization of the two forms. Thus the overall deficiency of glucuronidation in the fetus and newborn could possibly be attributed not to a total absence of UDPGT but to a deficiency of the high affinity form of GT coupled with a low activity of the low affinity

r*=0.64

pM

form. The latter was estimated to be 20-30 times lower than the adult value, a comparison made between the GT activity during the 12 days of culture with the average adult GT value determined in the crude homogenate. There was a progressive decline of GT activity during culture (Fig. 6), a pattern reminescent of that observed in our studies on sulfate conjugation of these cells (10). The next question to address would be to what extent each of these GT forms operates under physiological conditions. In man, bilirubin and a number of drugs such as morphine, oxazepam, lorazepam, and diflunisal are known to be conjugated almost exclusively with glucuranic acid (19-22). Clearly, the prevailing concentration of the acceptor substrate would, in a way, determine the participation of the high and/or low affinity forms of GT. It may be surmised that both low and high plasma concentrations of a drug may be encountered following its administration and thus the high and low affinity GT forms would be solicited to perform their functions. ACKNOWLEDGMENTS We thank the National University research scholarship to Miss Theresa search grant (RP 870349).

of Singapore for the award of a Tan May Chin and for the re-

REFERENCES 2

3

4

5

6

7

6

9

10

11

12

13

Days of Culture FIG. 6. Activity of UDPGT in extracts of human fetal liver cells during culture. Days 9 to 12 represent the confluent stage of culture.

1. Mulder, G. J., and Hagedoorn, 23,2101-2109. 2. Sundheimer, 11,433-440.

D. W., and Brendel,

A. H. (1974) K. (1983)

Biochem.

Pharmacol.

Drug Metab. D&OS.

50

TAN,

SIT,

3. Mulder, G. J., Weitering, J. G., Scholtens, E., Dawson, Pang, K. S. (1984) Biochem. Pharmacol. 33,3081-3087.

J. R., and

4. Wong, 4003.

31,4001-

K. P., and Yeo, T. (1982)

B&hem.

Phurmncol.

5. Slotkin, T., and DiStefano, V. (1970) Biochem. Phurmacol. 19, 125-131. 6. Wong, K. P., and Sourkes, T. L. (1967) And. Biochem. 21,444453. 7. Pang, K. S., Koster, H., Halsema, I. C. M., Scholtens, E., and Mulder, G. J. (1981) J. Phurmncol. Exp. Ther. 219,134-140. 8. Ching, M. S., Mihaly, G. W., Angus, P. W., Anderson, J. D., and Smallwood, R. A. (1986) J. Chromutogr. 380,190-195. 9. Wong, K. P. (1971) B&hem. J. 125,27-35. 10. Tan, T. M. C., Sit, K. H., and Wong, K. P. (1988) macol. 37,4629-4633. 11. Wong, K. P. (1970) Anal. B&hem. 35,35-45. 12. Lowry, (1951)

0. H., Rosebrough, N. J., Farr, J. Biol. Chem. 193,265-275.

13. Williams, P. A. (1985) The Netherlands.

Enxpack,

Version

Biochem.

A. L., and Randall, 2.0, Elsevier

Phar-

R. L. Science,

AND

WONG

14. Miners, Birkett, 15. Irshaid, 34.

J. O., Lillywhite, K. J., Matthews, D. J. (1988) Biochem. Phurmucol. Y. M., and Tephly, T. R. (1987)

16. Miners, J. O., Lillywhite, Phurmucol. 37.2839-2845.

K. J., and Birkett,

17. Dragacci, S., Hamar-Hansen, C., Magdalou, J., and Siest, 3923-3927. 18. Steiner, E., von Ther. 5,14-20. 19. Pacifici, G. M., Clin. Phurmucol. 20. Sawe, (1982)

Bahr,

A. P., Jones, M. E., and 37,665671. Mol. Pharmncol. 31,27D. J. (1988)

C., Fournel-Gigleux, G. (1987) Biochem.

C., and Rane,

Sawe, J., Kager, 22,553-558.

A. (1982)

L., and Rane,

J., Pacifici, G. M., Kager, L., von Acta Anaesth. &and. 74,47-51.

Bahr,

B&hem.

S., Lafaurie, Pharmacol. 36, Dev.

Pharmucol.

A. (1982)

Eur.

J.

C., and Rane,

A.

21. Greenblatt, D. J., Schillings, R. T., Kyriakopoulos, R. I., Sisenwine, S. F., Knowles, J. A., and Ruelius, Clin. Phurmucol. Ther. 20,329-341.

A. A., Shader, H. W. (1976)

22. Loewen, G. R., Herman, R. J., Ross, S. G., and Verbeeck, (1988) Brit. J. Clin. Pharmacol. 26,31-39.

R. K.