Biliary metabolites of all-trans-retinoic acid in the rat

ARCHNES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 334, No. 1, July 1, pp. 13-18, 1933

Biiiary Metabolites KEVIN

of All-tram-Retinoic

L. SKARE2

HECTOR

AND

Acid in the Rat’ F. DELUCA3

Department of Biochemistry, CoL!xge of Agricultural and Life Science-s, University Wisconsin-Madison, 420 Henry Mall, Mad&m, Wisumsin 53706 Received

November

13, 1982, and in revised

form

February

of

34, 1983

Biliary metabolites from physiological doses of all-trans-[10-3H]retinoic acid were examined in normal and vitamin A-deficient rats. The bile from normal and vitamin A-deficient rats contained approximately 60% of the administered dose following a 24h collection period. However, vitamin A-deficient rats show a 6-h delay in the excretion of radioactivity compared to normal rats. Retinoyl-/3-glucuronide excretion was particularly sensitive to the vitamin A status of the rats. In normal rats, retinoyl-& glucuronide reached a maximum concentration of 235 pmol/ml of bile 2 h following the dose and then rapidly declined. Vitamin A-deficient rats show a relatively constant concentration of this metabolite (100-150 pmol/ml of bile) over a 10-h collection period. Retinoic acid excretion was low in both normal and deficient rats. The concentration of retinotaurine, a recently identified biliary metabolite, was approximately equal to retinoyl-fl-glucuronide in normal rats and appeared in the bile 2 h later than the glucuronide.

The metabolism of retinoic acid to polar metabolites has been an area of great interest for the past two decades. Retinoic acid is known to support the growth of vitamin A-deficient animals, to maintain the differentiation of epithelial tissues, and to suppress neoplastic transformation (l3). However, retinoic acid cannot support the reproductive (4) or visual (1) functions of retinol. The possibility of the biotransformation of retinoic acid to more “active” forms has prompted a reinvestigation into the identity of the polar metabolites of retinoic acid.

Retinoic acid is known to be rapidly cleared from tissues and blood and subsequently appears as polar metabolites in the bile of animals dosed with either pharmacological or physiological levels of retinoic acid (5, 6, 8). Three polar metabolites have now been isolated and identified from the bile of rats dosed with retinoic acid. Retinoyl-/3-glucuronide (6-8) and retinotaurine (10) were identified after the administration of all-trans-retinoic acid and 13-cis-4-ketoretinoyl+glucuronide was identified after the administration of 13-cti-retinoic acid (9). Recently, 13&s- and all-trans-retinoyl-fi-glucuronides have been detected in the intestines of bile duct cannulated rats (11) and in several other organs (12). Hence, its site of synthesis does not appear to be restricted to the liver. This paper examines the metabolites of retinoic acid as a function of time following a physiological dose of all-trans-retinoic acid given to either normal or vitamin A-deficient rats.

1 This work was supported by program project Grant AM-14331 from the National Institutes of Health, a predoctoral fellowship from Procter and Gamble, and by the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation. ‘Present address: Procter and Gamble Co., Research and Development Department, Miami Valley Laboratories, P.O. Box 39175, Cincinnati, Ohio 4524’7. a Author to whom correspondence should be addressed. 13

0003-986w33 Copyright All rights

$3.00

8 1983 by Academic Press, Inc. of reproduction in any form reserved.

14

SKARE MATERIALS

AND

AND

METHODS

General procedures. HPLC was performed with a microprocessor-controlled Beckman Model 332 Gradient Liquid Chromatograph (Beckman Instruments, Irvine, Calif.) equipped with a Waters Model 440 absorbance detector operating at 340 nm and a Waters U6K universal injector (Waters Inc., Milford, Mass.). HPLC-grade solvents from Fisher Chemical Co. (Chicago, Ill.) were used with the exception of water which was glass distilled before use and run through 0.2rrn filters (Millipore Corp., Bedford, Mass.). HPLC separations were performed using a reverse-phase PBondapak C-18 column (4 X 300 mm) in series with a Whatman guard column (2.3 X 70 mm) packed with a Bondapak C-lS/Porasil B resin (Waters Associates). For gradient elutions, the system was equilibrated with methanol:water (2:98) and then eluted with a 2-h linear gradient from methanol:water (298) to 100% methanol. All solvents used for HPLC contained 10 mM ammonium acetate to minimize tailing. The elution position of eight retinoid standards (listed under Chemicals) was monitored by uv absorbance at 340 nm. These compounds served as internal standards in all gradient elutions. This system gives baseline resolution of all eight retinoids. For quantitation of retinotaurine in bile samples, a 4.6 X 250-mm Zorbax-ODS’ column (DuPont, Inc., Wilmington, Del.) eluted with methanol:water (35:65) was used, as described previously (10). Radioactivity was determined by liquid scintillation counting using a Packard Prias PLD Tri-Carb minivial counter (Packard Instruments, Downers Grove, Ill.). All samples (1.1 ml) were counted in 3a70B scintillation fluid (Research Products Intl., Elk Grove Village, Ill.). For quantitation of metabolites in bile samples, counting efficiencies were determined automatically using a programmable quench curve stored in the memory of the microprocessor. For specific quantitation of retinoyl+‘-glucuronide, retinotaurine, and retinoic acid, 50- to lOO-~1 aliquots of each 2-h bile sample were injected directly onto a reverse-phase column as described previously (6). Chromatographic conditions are as described above. Whole fractions (1.1 ml) were counted and absolute amounts were calculated from the radioactivity present under the respective peaks and the specific activity of the administered dose. For comparison purposes, the amount of retinoyl+-glucuronide, retinotaurine, and all-trans-retinoic acid were normalized to yield results expressed as picomoles per milliliter of bile. Chewkuls. Nonradioactive all-trans-retinoic acid and retinol were purchased from Eastman Organic Chemicals (Rochester, N. Y.) and did not require fur-

* Abbreviations used: ODS, octadecylsilane; dimethyl sulfoxide.

Me,?&,

DE

LUCA

ther purification. The nonradioactive retinoids which were used as internal standards for HPLC were generous gifts from the Hoffmann-La Roche, Inc. (Nutley, N. J.). These standards are: l-hydroxymethyl-4-ketoretinoic acid,54-ketoretinoic acid, 4-hydroxyretinoic acid, 5,6-epoxyretinoic acid, and 13-c& retinoic acid. Methyl retinoate was prepared from all-trans-retinoic acid by treatment with diazomethane. Sodium chloride, dimethylsulfoxide (Me$O), and glucose were obtained from Sigma Chemical Company (St. Louis, MO.). Sodium pentobarbital (Nembutal) was obtained from Abbott Laboratories (Chicago, Ill.). HPLC-grade ammonium acetate was purchased from Fisher Chemical Company (Itasca, Ill.). PutiJcation of radiochemicals. All-trans-[10-3H]retinoic acid (sp act 2.8 Ci/mmol) was a gift from Hoffmann-La Roche. It was purified before use on a 4 X300-mm PBondapak C-18 column eluted with methanol:water (6634) containing 10 m?d ammonium acetate. The all-truns-retinoic acid peak elutes between 25 and 35 ml using this system. Following purification, the compounds were 94% all-trans-retinoic acid and 4% 13-cis-retinoic acid. This is considered radiochemically pure, since on rechromatography of 100% all-trans-radiolabeled retinoic acid the same distribution (i.e., 94% trans, 4% cis, and 2% loss) is observed using the above-described system. Animals. Male rats were purchased from the Holtzman Company (Madison, Wise.) and housed in overhanging wire cages with free access to food and water. One group of six rats was made vitamin A-deficient using a chemically defined vitamin A-free diet previously described (13). The deficient rats were used for experiments after approximately 6 to 7 weeks on the deficient diet. Normal rats were maintained on a pelleted stock diet until needed (Wayne Lab-Blox, Allied Mills, Inc., Chicago, Ill.). In the case of all data, representative results for one animal are presented. However, six samples were analyzed in each case and the data are within 20% of each other. Bile duct cannubtions. All rats were anesthetized by intrajugular injection of Nembutal(25 mg/kg body wt). These injections were given under light ether anesthesia. The bile ducts were then cannulated using No. 10 polyethylene tubing. The abdominal incision was closed with sutures and metal clips after bile flow was established. The flow of bile was similar in all groups or 0.7 ml/h. The rats were then injected intrajugularly with 2 pg of all-truns-[10-3H]retinoic acid. All injections and subsequent bile collections were performed under yellow lights to minimize isomerization. Following the injection of labeled retinoic acid, all rats were placed in restraining cages ‘This mediate identified

compound has been proposed as an interin the biosynthesis of retinotaurine, a newly polar biliary metabolite.

METABOLITES

OF

ALL-truns-RETINOIC

15

ACID

and allowed a 10% solution of glucose ad Z&turn Bile was collected every 2 h and stored in minivials, under nitrogen, at -20°C until analyzed by HPLC. As described above, aliquots of each bile sample were directly analyzed by HPLC with no prior purification. RESULTS

Time course of appearance of radioactivity in the bile of rats given a physiological dose of all-truns-retinoic acid is shown in Fig. 1. The data presented are from a single rat but are representative of the six animals per group studied. A comparison of the cumulative recovery of radioactivity for normal rats (Fig. 1A) and vitamin A-deficient rats (Fig. 1B) shows that both groups contain approximately 60% of the dose in the bile following a physiological dose. However, the kinetics for excretion differ between these two groups. Normal rats have 30% of the dose

Hours

After

Injection

2 100,

,100

3 8. f760-

4

-60zL ;:

6

12

16

20

FIG. 1. Time course for the excretion of radioactivity into the bile of rats following intrajugular injections of (A) 2 pg of all-trum-[10-3H]retinoic acid in normal rats; (B) 2 pg of all-trons-[lO-aH]retinoic acid in deficient rats. Open circles (0) represent cumulative recovery of radioactivity in the bile and closed circles (0) represent the percentage of dose excreted per 2-h collection period.

Fmction

No. (I. I ml I

FIG. 2. Reverse-phase HPLC of bile from normal rats given an intrajugular injection of 2 *g of alltrons-[10-3H]retinoic acid. Bile samples were analyzed: O-2, 2-4, 4-6, and 6-8 h. Only the 0- to 2-b profile is shown. The column was developed using a 2-h linear gradient from methanol:water (2:98) to 100% methanol as described under Materials and Methods. Vertical arrows indicate the elution position of the retinoids used as internal standards. These standards are: (a) 1-hydroxymethyl-4-ketoretinoic acid, (b) I-ketoretinoic acid, (c) I-hydroxyretinoic acid, (d) 5,6-epoxyretinoic acid, (e) 13-cis-retinoic acid, (f) all-trans-retinoic acid, (g) retinol, and (h) methyl retinoate. Peak 7 represents both 13-c& and all-trunsretinoyl-b-glucuronide.

in the bile after 3 h of collection (Fig. lA), whereas deficient rats require 9 h of collection before 30% of the dose is present in the bile (Fig. 1B). When the percentage of the dose recovered per 2-h interval is examined for normal rats (Fig. lA), one can see that the radioactivity reaches a maximum at 4 h and then rapidly declines. Vitamin A-deficient rats, on the other hand, do not reach a maximum until approximately 11 h and then decline more slowly (Fig. 1B). The metabolites found in a representative bile sample are shown in the Fig. 2. This metabolite profile was obtained by subjecting aliquots of 2-h bile samples to HPLC analysis as described under Materials and Methods. The elution positions of the eight internal standards are marked by vertical arrows. The radioactive peaks from these profiles are designated by the numbers 3 through 8. Each peak may contain more than one biliary metabolite due to the limited resolution by the gradient system. The peak 5 region is known to contain l-hydroxymethyl-4-ketoretinoic acid and retinotaurine. Peak ‘7 corresponds to l&c&+ and all-truns-retinoyl+glucuro-

16

SKARE

AND

nide and peak 8 is all-trans-retinoic acid. The 13-c&- and all-trans-retinoyl-@-glucuronide were identified by hydrolysis with P-glucuronidase followed by chromatography on the same HPLC system as described in earlier publications (6, 8, 14). Figure 2 and Table I show that retinoyl@-glucuronide is the major metabolite in the 0- to 2-h bile sample. Retinoic acid is at its maximum concentration during this time period but only represents approximately 3% of the administered radioactivity. As observed by others (6,14), polar metabolites represent a major portion of the biliary radioactivity even at early times following the dose. A radioactive peak which comigrates with the l-hydroxymethyl-4-keto-retinoic acid standard is seen in Fig. 2. However, the presence of this metabolite in bile cannot be confirmed until it is isolated and identified. No radioactive peaks which comigrate with standard 5,6-epoxyretinoic acid, 4-hydroxyretinoic acid, or 4-ketoretinoic acid were observed in these metabolic profiles. Table I shows that in the 2- to 4-h bile sample from normal rats, the all-transretinoic acid peak has disappeared and the retinoyl-8-glucuronide peak has decreased in concentration from 235 to 110 pmol/ml TABLE METABOLITES

DE

LUCA

of bile. Concomitant with these changes are increases in the concentration of the polar metabolites present in peaks 3-5. By 4-6 h all peaks have declined. The three polar peaks (3-5) were still at a concentration above 100 pmol/ml of bile. The most polar metabolites predominate at the later collection times. Although retinoyl-& glucuronide and peak 6 are still present in 6- to 8-h bile, the polar metabolites (peaks 3-5) account for the majority of the radioactivity. The radioactivity at the solvent front also increases with time of collection. Peak 3 reaches a concentration of 480 pmol/ml of bile, whereas retinoyl-& glucuronide only reaches a concentration of 235 pmol/ml of bile. A very different pattern of biliary excretion is seen for vitamin A-deficient rats (Table I). The appearance of radioactivity in the bile of vitamin A-deficient rats is delayed compared to normal rats (Fig. 1B). At early time points, the polar metabolites are much lower in concentration in vitamin A-deficient rats than in normal rats (Table I). The largest single metabolite in the 2- to 4-h bile sample is retinoyl-&glucuronide which accounts for 29% of the total biliary radioactivity. A small amount of all-trans-retinoic acid is excreted durI

IN BILE OF NORMAL AND VITAMIN WITH ALL-~~cw-[~O-~H]RETINOIC

A-DEFICIENT ACID

Metabolites Collection

bile

period 3

4

5

6

180 480 180 100

140 300 120 90

198 500 150 65

150 120 35 20

-

20 35 110 150

20 25 85 100

00 Normal o-2 2-4 4-6 6-8 Vitamin o-2 2-4 4-6 6-8 9-10

in pmoI/ml

RATS INJECTED

Glucuronide

Note. The above within each group

Retinoic

acid

235 110 35 10

100 210 50 20

-

-

-

-

20 20 80 110

110 120 140 100

10 15 40 50

4 4 4 2

A deficient 40 62 215 295

Retinotaurine

results are representative of data obtained are within 20% of each other.

from

six rats

in each group.

Values

from

rats

METABOLITES

OF

ALL-trans-RETINOIC

ing this time and the major polar metabolites are in peak 3. The 4- to 6-h bile sample shows a metabolic profile very similar to that seen in the 2- to 4-h bile sample. The concentrations of retinoyl-P-glucuronide and all-trans-retinoic acid are higher in deficient rats than in normal rats following 8 h of collection. At times up to 6 h of collection, deficient rats appear to convert more of the available retinoic acid to retinoyl+?-glucuronide rather than polar metabolites (peaks 3-6). Retinoic acid and retinoyl+glucuronide are still present in 8- to 10-h bile. At these later collection times, the very polar metabolites begin to account for a large percentage of the excreted radioactivity however. In particular, peak 3 rises in concentration rapidly following 8 h of collection. It is of some interest that the excretion of retinoic acid remains relatively constant throughout the first 10 h of collection in the deficient rats whereas normal rats only excrete retinoic acid during the first 2 h. The concentration of retinoyl-gglucuronide is elevated at all times examined in deficient rats but declines rapidly after only 2 h in normal rats. Although the concentration of retinoyl/3-glucuronide is approximately twice that of retinotaurine in the 0- to 2-h bile sample, at 2- to 4-h retinotaurine is approximately twice the glucuronide concentration. There is also a delay in the appearance of other polar metabolites compared to retinoyl-@-glucuronide. In spite of the different kinetics observed for these two identified metabolites, both comprise approximately lo-12% of the total biliary radioactivity after a 24-h collection period (8, 10). DISCUSSION

Several investigators have shown that retinoic acid rapidly disappears from the blood and tissues of rats following the administration of either the all-truns- or 13&-form (5, 6, 18). Concomitantly, there is a rapid excretion of polar metabolites of retinoic acid into the bile (6,8,9). The results presented in this paper confirm the

ACID

17

rapid excretion of polar metabolites into bile. However, the vitamin A status of the rat is a determining factor in the rate of excretion of these polar metabolites when physiologic doses are used. When physiological doses of retinoic acid are administered to rats, a delay of 4-6 h is observed for the appearance of metabolites in the bile of vitamin A-deficient rats compared to normal rats. As discussed earlier, this delay may result from the loss of retinoid-induced enzymes normally used to metabolize retinoic acid to more polar metabolites. Indeed, recent work has shown that the vitamin A status of animals is important for the conversion of retinoic acid to more polar metabolites (15). These authors demonstrated that prior induction of vitamin A-deficient rats with retinoic acid allows for the conversion of retinoic acid to 4-hydroxyretinoic acid. An alternative explanation for the delay in excretion is that retinoic acid may be transported to target organs in need of vitamin A. Thus, metabolites may not reach the liver and be excreted into the bile until later times. Polar metabolites may also require longer times for excretion if several enzymatic steps are required for the biosynthesis of these metabolites. RetinoylP-glucuronide achieves maximum concentrations 2 h earlier than retinotaurine. Since retinotaurine biosynthesis requires at least six steps whereas retinoyl-&glucuronide only requires one, a delay in retinotaurine excretion might be expected. The excretion kinetics for retinoyl-& glucuronide in normal rats are dramatically different from those observed for vitamin A-deficient rats following a physiological dose. Deficient rats show a relatively constant excretion of retinoyl-flglucuronide over the first 11 h of collection, whereas normal rats show a rapid rise followed by a sharp decline. The reason for this difference is unknown but it may represent an important regulatory step in the metabolism of retinoic acid. For example, the enterohepatic circulation of retinoyl@glucuronide in the vitamin A-deficient rat may return retinoic acid in the portal circulation for further use. A radioactive peak comigrating with l-

18

SKARE

AND

hydroxymethyl-4-ketoretinoic acid was observed in normal rats up to 4 h of collection and in deficient rats up to 10 h of collection. Rietz et ah (16) have identified this metabolite in the urine of rats injected with 27.5 mg of retinoic acid. No detectable peaks comigrating with 4-hydroxyretinoic acid, 5,6-epoxyretinoic acid, or methyl retinoate were observed in rats dosed with physiological levels of retinoic acid. There are, however, radioactive compounds which migrate very close to 4-ketoretinoic acid and may correspond to cis isomers of this metabolite. The synthesis of 4-hydroxy- and 4-ketoretinoic acid is known to take place in liver microsomes (17) but apparently these metabolites are not excreted in bile. One possible explanation for this observation is that these two metabolites may be conjugated in the liver prior to excretion in the bile. This is a very likely possibility since 13-cis-4-ketoretinoyl-fl-glucuronide has recently been isolated from the bile of normal rats injected with approximately 5 pg of 13-c&retinoic acid (9). These authors found that rats dosed with either 13-cis- or all-transretinoic acid excrete approximately the same amount of radioactivity within 24 h. However, more of the all-truns isomer is metabolized to polar compounds. Several of the polar metabolites were sensitive to incubation with B-glucuronidase consistent with the hypothesis that many of the polar metabolites are conjugated in the liver prior to excretion in bile. The study mentioned above demonstrates that biliary excretion of metabolites is dependent on the isomer injected in addition to the vitamin A status of the rats as reported in this paper.

DE

LUCA REFERENCES

1. DOWLING, J. E., AND WALD, G. (1960) Proc Nat. Acad. Sci. USA 46,587-608. 2. ZILE, M., AND DELUCA, H. F. (1968) J. N&r. 94, 302-308. 3. SPORN, M. B., DUNLOP, N. M., NEWTON, D. L., AND SMITH, J. M. (1976) Fed Proc. 35,1332-1338. 4. THOMPSON, J. N., HOWELL, J. McC., AND PITT, G. A. J. (1964) Proc. R. Sot. Lmw!m Ser. B 159, 510-535. 5. ZACHMAN, R. D., DUNAGIN, P. E., AND OLSON, J. A. (1966) .I Lipid Res. 7,3-9. 6. SWANSON, B. N., FROLIK, C. A., ZAHAREVITZ, D. W., ROLLER, P. P., AND SPORN, M. B. (1981) B&hem Pharmacol 30,107-113. 7. DUNAGIN, P. E., MEADOWS, E. H., AND OLSON, J. A. (1965) Science 148,86-87. 8. ZILE, M. H., SCHNOES, H. K., AND DELUCA, H. F. (1980) Proc. Nat. Acad. Sci USA 77,3230-3233. 9. FROLIK, C. A., SWANSON, B. N., DART, L. L., AND SPORN, M. B. (1981) Arch. Biochem Biophys. 208,344-352. 10. SKARE, K. L., SCHNOES, H. K., AND DELUCA, H. F. (1982) Biochemistry, 21, 3308-3317. 11. ZILE, M. H., INHORN, R. C., AND DELUCA, H. F. (1982) J. Biol. Chem 257, 3537-3543. 12. SILVA, D. P., AND DELUCA, H. F. (1982) Biochxm. J. 206, 33-41. 13. DELUCA, H. F., MANATT, M. R., MADSEN, N., AND OLSON, E. B. (1963) .I Nutr. 81, 383-386. 14. ZILE, M. H., INHORN, R. C., AND DELUCA, (1982) J. Biol Chem. 257, 3544-3550.

H. F.

15. ROBERTS, A. B., FROLIK, C. A., NICHOLS, M. D., AND SPORN, M. B. (1979) J. Bill Chem 254, 6303-6309. 16. RIETZ, P., WISS, O., AND WEBER, F. (1974) in Vitamins and Hormones, (Harris, R. S., Diczfalusy, E., Munson, P. L., and Glover, J., eds.), pp. 237-249, Academic Press, New York. 17. ROBERTS, A. B., LAMB, (1980) Arch B&hem

L. C., AND SPORN, M. B. Biophgs. 199,374-383.

18. WANG, C. C., HODGES, R. E., JR., AND HILL, (1978) Anal B&hem 89, 220-224.

D. L.