Acta pharmacol. et toxicol. 1976, 39, 419-432.

From the Department of Pharmacology, University of Bergen, MFH-Bygget, 5016 Haukeland sykehus, Bergen, Norway

The Metabolism of Biphenyl. 11. Phenolic Metabolites in the Rat BY Trygve Meyer’ and Ronald R. Scheline (Received February 6, 1976; Accepted February 20, 1976)

A bstmct: The metabolic conversion of biphenyl to phenolic metabolites was studied in the rat. The metabolites were identified by mass spectrometry and quantified by gas chromatography following conversion to their trimethylsilyl (TMS) ether\. The main route of excretion was via the urine, and the major part (22.3 %) of the biphenyl metabolites was excreted in the first 24 hrs. The total urinary recovery 96 hrs after administration was 29.5 % of the dose and the metabolites detected were conjugates of mono-, di- and tri-hydroxy derivatives o f biphenyl as well as the meta- and para-methyl ethers of the catecholic compounds. The two main urinary metabolites were 4-hydroxybiphenyl (7.7 76) and 4,4‘-dihydroxybiphenyl (11.4 %). The experiments also showed that biphenyl has to be hydroxylated and then conjugated before it appears in the rat bile. Thus, 5.2 % of the dose was found in the 24 hrs bile as conjugates, mainly of 4-hydroxy-, 4,4’-dihydroxy- and 3,4,4’-trihydroxy-biphenyl. Faecal excretion of phenolic biphenyl derivatives was found to be of minor importance, but 4.7 70 of the dose was detected during the first 24 hrs after dosing. The following previously undetected metabolites of biphenyl were found: 3,4‘dihydroxy-, 3,4,4’-trihydroxy-, 3,4’-dihydroxy-4-methoxy-and 4,4’-dihydroxy-3rnethoxy-biphenyl. Kry-words: Biphenyl - metabolism - phenols

-

rat.

Metabolic experiments with biphenyl in several laboratory animal species have revealed a series of phenolic metabolites. An early report by KLINGENBERG (189 1) indicated that biphenyl was converted into 4-hydroxybiphenyl in the dog. The same metabolic conversion has been demonstrated in the rabbit (STROUD1940; BLOCK& CORNISH1959). 1

Present address: National Institute of Forensic Toxicology, Oslo, Norway.

420

T. MEYER AND R. R. SCHELINE

Similar metabolic experiments with biphenyl in the rat revealed several metabolites of which 4-hydroxy-, 4,4'-dihydroxy- and 3,4-dihydroxy-biphenyl were isolated and identified by WESTet al. (1953, 1955 & 1956). Later, the use of gas chromatography has allowed the identification of further phenolic metabolites of biphenyl. Thus, 2-hydroxybiphenyl as well as the previously known 4-hydroxy-, 3,4-dihydroxy- and 4,4'-dihydroxyderivatives were established as metabolites of biphenyl in the rabbit (RAIG & AMMON1970). Subsequently, 3-hydroxybiphenyl and the two monomethylated derivatives of 3,4-dihydroxybiphenyl were also shown to be formed in this species (RAE & AMMON 1972). In experiments with I4C-biphenyl MEYERet al. (1976) were able to show both an acidic and a phenolic urinary fraction in rats. The purpose of the present investigation was to study the phenolic fraction from both a qualitative and quantitative point of view.

Materials and Methods Cherniccrlu. Biphenyl (m. p. 6849") was a gift from the Department of Chemistry, University of Bergen, Norway. It was found to be gas chromatographically pure. 2-Hydroxybiphenyl, zone melted (m. p. 57-58') and 4,4'-dihydroxybiphenyl (m. p. 271-273") were purchased for EGA-Chemie KG, W. Germany. 3-Hydroxybiphenyl (m. p. 78-79") and 4-hydroxybiphenyl (m. p. 165-166") were purchased from K & K Laboratories, Inc., U. S. A. 2,2'-Dihydroxybiphenyl (m. p. 107-108') was purchased from Koch-Light Laboratories, Ltd., England. 3,4-Dihydroxybiphenyl (m. p. 137-138") was generously provided by Professor L. Horner, Mainz, W. Germany. 4-Hydroxy-3metboxybiphenyl (m. p. 67-69') was prepared by the method of RAIG & AMMON(1972). 3,4,4'-Trihydroxybiphenyl (m.p. 240") was prepared by the method described by HORNER& WEBER (1967). 2,5-DihydroxybiphenyI was prepared by reduction of 2-phenyl-Q-benzoquinone purchased from Aldrich Chemical Co., Inc., U. S. A., and the crude product was silylated with Tri-Sil Pierce Chemical Co., U. S. A,) directly to give the TMS-ether. The purity of all compounds was suitable for the intended purpose. The melting points are uncorrected. Atiinial experiments.

Male albino rats weighing 200-300 g were used in all experiments. The animals were maintained on a commercial pellet diet (Vesterlandske Kjgjpelag, Bergen) both before and during the experiments, and they were given both diet and drinking water ad libitum. The test compounds were given as a solution in soya oil (1 ml) by stomach tube or in a few cases by intracaecal'injection, and the doses used were 100 and in some cases 400 mg/kg. Urine and faeces were collected separately for 24 hrs periods in containers maintained at 0". In some experiments the animals were given a daily dose of 200 mg neomycin sulphate in 2 ml distilled water for three days before the administration of the test compound and during the experimental period.

PHENOLIC BIPHENYL METABOLITES IN RAT

42 1

Bile samples were collected by inserting a thin plastic tube in the common bile duct (pentobarbitone (mebumalum NFN) anaesthesia) (SCHELINE1968) before oral administration of the test compound. Biliary excretion was prevented by surgically ligating (pentobarbitone anaesthesia) the common bile duct at two points and severing the duct between these two points. Extracrion o f urinary nzetnbolites. The 24 hrs urine samples, after thawing and filtering, were diluted to 25 ml with distilled water before being acidified to pH 2-3 and extracted five times with 25 ml portions of ether. The combined ether extracts were shaken three times with 25 ml portions of 5 "/o aq. NaHCO, and once with 0.1 N-HCI before being dried over anhydr. Na,SO, and evaporated t o dryness. The residues contained the free phenolic fraction of the urine and were dissolved in 1 ml Tri-Sil. The water residues after ether extractions were adjusted to pH 5 with 1 N-NaOH and acetate buffer and then hydrolyzed with a preparation containing f&glucuronidase and sulphatase (glusulase, Endo Laboratories, U. S. A.) according to the method used of RAKKE & SCHELINE (1969) before being extracted and treated as described above. This gave the bound phenolic fraction of the urine. Extraction of biliary metabolites. The 6-7 and 24 hrs bile samples were first treated with glusulase and then extracted with ether as described above. Extraction o f faecal metabolites. The faecal samples were mixed with 25 ml distilled water in a Servall Omni-mixer, the pH adjusted to 5 with 0.1 N-HCI and acetate buffer and then left overnight at 37". The samples were then adjusted to pH 2 with 1 N-HC1 and ether extracted continuously overnight. Quuntitative determiriation of metabolites. The approximate amounts of 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl as TMSethers were estimated from the urine samples from rats given biphenyl as described above. Standard solutions containing these compounds in various concentrations, including the estimated concentrations in 1 ml Tri-Sil were made and the gas chromatographic areas were plotted against the respective concentrations. The resulting plots were linear. The same amounts of the two compounds were added to urine samples from untreated rats. After ether extractions as described above, the residues were dissolved in 1 ml Tri-Sil and the gas chromatographic areas were again plotted against the respective concentrations. This gave approximately the same plots as described above and the extraction of the phenolic metabolites from rat urine was therefore assumed to he essentially quantitative. The calibration runs were made with a solution containing known amounts of each reference compound. The response factor (amountlarea) and retention time (in min.) for each compound were stored in the memory of the integrating unit before analysis of the biological samples. The amounts of each metabolite found are based upon the calibration of the integrator unit. Metabolites for which no reference compound was available were quantified by means of known response factors of similar compounds. Fig. 1 shows a gas chromatogram of the first day phenolic fraction from rat urine after silylation.

T. MEYER AND R. R. SCHELINE

422

Incubation experiments. Enzyme preparation and incubations of 3,4-dihydroxybiphenyl and 3,4,4'-trihydroxybiphenyl with the enzyme catechol-0-methyl transferase (COMT) from rat liver were carried out according to the method described by AXELROD & TOMCHICK (1958). Incubation of 3,4,4'-trihydroxybiphenyl under anaerobic conditions with rat caecal microorganisms was carried out as described previously (SCHELINE 1966 & 1968). The in vitro samples were acidified and extracted with ether as described above. Gas chromatography. The samples containing the TMS-ethers of the metabolites were analysed on a Hewlett-Packard gas chromatograph Model 5710 A equipped with flame ionization detector. The glass column was 0.3 (int. diam.) X 145 cm and was packed with 3 "70 OV-1 on Chromosorb Q (8&100 mesh). The temperature was programmed from 80270" a t 6.3"lmin. and argon (30 mllmin.) was used as carrier gas. The capillary analysis was performed on the above mentioned gas chromatograph using a splitless SGE. injector and a 0.37 mm (int. diam.) X 20 m OV-1 Grob glass column (H. G. Jaeggi, Switzerland). The temperature was programmed from 100-250" at 6.3"imin. and helium (0.5 kg pressure) was used as carrier gas. The recording integrator used was a Hewlett-Packard Model 3380 A.

+

Mass spectrometry. The silylated samples and the appropriate standards were analysed by combined gas chromatography/mass spectrometry (Varian MAT 111) using a 0.2 (int. diam.) X 120 cm glass column containing 3 96 OV-1 on Gas-Chrom Q (100-120 mesh). The column temperature was programmed from 80-270" a t 6"lmin. and helium (15 mllmin.) was used as carrier gas.

1

Fig. 1. A typical gas chromatogram of a silylated first day urine sample showing the phenolic metabolites of biphenyl in the rat.

423

PHENOLIC BIPHENYL METABOLITES IN RAT

Table 1. Retention times and relative abundance of M+ and prominent fragments of biphenyl and biphenyl derivatives (as TMS-ethers). Retention time (min.) M+ Prominent fragments (rn/e)

Compound number

Chemical name

~~

Relative abundance

Capillary

ov-1 ov-1

1 Biphenyl

2

3 70

2-Hydrouybiphenyl

8.26

4.55

154 100

153 44

152 31

79 19

77 25

7’92

242 33

227 61

211 100

152 17

106

12.15

14S0

10‘14

242 86

227 100

211 76

152 14

113.5 24

242

85

227 100

211 65

152 24

113.5 41

272 42 272 41

242 100 257 14

212 15 242 100

121 15 121 16

113.5 27 113.5 23

330

315 15

242 15

227 9

211 12

330

315 65

165 7

150 42

142 8

330 100

315 34

165 4

150 34

142 4

22.97

360 73 360 18‘95 83

330 100 330 100

158 39 150 56

1.50 48 147 74

142 22 93 67

418 23.74

19.72

403 5

330 315 4 2 1

165 2

330 “*”

I2’O2

315 53

300 79

142 32

17

~

3

3-Hydrouybiphenyl

4

4-Hydroxybiphenyl 3-Hydroxy-4-methoxy biphenyl:li 4-Hydroxy-3-methoxybiphenY1

17.41

l3’Oo

17.41

13”’

6

3,4-DihydroxybiphenyI

18.66

14.32

7

3,4’-Dihydroxybiphenyla)

20.36

16.15

8

4,4’-Dihydroxybiphenyl

21.10

16.98

22.97

18.70

3,4’-Dihydroxy-4-methoxybiphenyl4 4,4-Dihydroxy-3-methoxybiphen yla)

10

3,4,4’-Trihydroxybiphenyla)

11 2,5-Dihydroxybiphenyl

a) No reference compound available (see text)

100

227 17

424

T. MEYER AND R. R. SCHELINE

Table 2. Normal metabolism and excretion of biphenyl in rats. Urine collected daily for 4 days and faeces collected for 24 hrs after oral administration of 25 mg (approximately 100 mg/kg) biphenyllanimal. Results are mean values as % dose, with numbers of animal in parenthesis. Faeces (3)

Urine (5)

+

Metabolite

Biphenyl 2-Hydroxybiphenyl 3-Hydrox ybiphen yl 4-Hydroxybiphenyl 2,5-DihydroxybiphenyI 3-Hydroxy-4-methoxybiphenyl ) 4-Hydroxy-3-methoxybiphenyl) 3,4-Dihydroxybiphenyl 3,4’-Di hydroxybipheny 1 4,4'-Dihydroxybiphen yl 3,4’-Dihydroxy-4-methoxybiphenyl) 4,4’-Dihydroxy-3-methoxybiphenyl )

3,4,4‘-Trihydroxybiphenyl Total

3. 1.day

2. day 4.day

Total

0.1 0.4 0.9 6.8 a)

0.1 0.5 0.4 0.7 b)

b) 0.1 0.3 0.2 b)

0.2 1.0 1.6 7.7

b) 0.3 0.5 1.o b)

0.1

b)

b)

0.1

b)

0.6

0.2 0.3 1.7

b)

0.8 2.6 11.4

b) b) 1.8

O”

b)

1.5 9.6 0*5 1.8 22.3

0.8 0.1

0‘3

1. day

0.9

0.5

3.2

1.1

5.1

2.1

29.5

4.7

a) Trace metabolite b) Not detected c) Combined quantitation

The mass spectra of the metabolites and the parent compound were taken at 80 eV and at a scan speed of 100 massesfsec.

Results The retention times and the relative abundance of M+ and the most prominent fragments of the reference compounds as T M S ethers are listed in table 1. Metabolites for which no reference compound was available were identified mainly on the fragmentation pattern shown in the mass spectrum but also in relevant biological experiments. Urinary metabolites. Unhydrolysed urine contained only trace amounts of free phenolic metabolites. Hence, the urines were mainly analysed as total urinary samples.

PHENOLIC BIPHENYL METABOLITES IN R A T

425

The metabolites detected in the 96 hrs urines of rats given a single oral dose of biphenyl are shown in table 2, from which it can be seen that the total amount accounted for was 29.5 70.No phenolic metabolites could be detected after this period. The major metabolites are 4-hydroxybiphenyl (7.7 70)and 4,4‘-dihydroxybiphenyl (13.4 %). In both cases, most of this material was excreted within 24 hrs. Several phenolic metabolites not previously reported were detected in the rat urines in these experiments. Thus, 3,4’-dihydroxybiphenyl amounted to 2.670 of the dose after 4 days. The identity of this metabolite was established by means of mass spectrometry and biological experiments. Both this metabolite and 4,4’-dihydroxybiphenyl showed very similar mass spectra (as TMS derivatives, table 1). This fact indicates that one hydroxyl group is located in each aromatic ring. It is reasonable to assume that one of these is located in the 4’-position. This was shown to be the case as the administration of 4-hydroxybiphenyl to rats resulted in the urinary excretion,

Table 3. Urinary metabolites from biphenyl in rats given neomycin. Urine collected daily for 4 days after oral administration of 25 mg (approximately 100 mglkg) biphenyllanimal. Results are mean values as % dose, with numbers of animals in parenthesis. Urine (2)

+

Metabolite

3. 1. day

Biphenyl 2-Hydroxybiphen y l 3-Hydroxybipheny l 4-Hydroxybiphenyl 2,s-Dih ydrax ybiphenyl

3-Hydroxy-4-methoxybiphenyl) 4-Hydroxy-3-methoxybiphenyl ) ‘) 3,4-Dihydroxybiphenyl 3,4’-Dihydroxybiphenyl 4,4’-Dihydroxybiphenyl 3,4’-Dihydroxy-4-methoxybiphenyI) 4,4’-Dihydroxy-3-methoxybiphenyl) ‘) 3,4,4’-Trihydroxybiphenyl Total a) Trace metabolite b) Not detected c) Combined quantitation

2. day

4. day

Total

b)

b)

b)

0.t 0.6

0.1 0.1

8.3 bl

0.9 bl

0.1 0.1 0.2 b)

0.1

4

b)

0.1

0.9 1.3 11.1

0.1 1.6

a) 0.1 0.1

1.0 1.6 12.8

0.4

0.1

a)

0.5

1.6

0.3

O.t

2.0

24.4

3.4

0.7

28.5

0.2

0.3

0.8 9.4

T.MEYER AND R. R. SCHELINE

426

as conjugates, of large amounts of unchanged compound and 4,4‘-dihydroxy biphenyl together with some of the unknown compound. When 3-hydroxybiphenyl was given, the urine contained large amounts of unchanged compound and the unknown dihydroxy isomer as well as small amounts of 3,4-dihydroxybiphenyl. These data indicate that the unknown is 3,4‘-dihydroxybiphenyl. The tendency of hydroxylating a free 4-position before the other positions is also encountered with the dihydroxybiphenyls. Thus, 3,4,4‘-trihydroxybiphenyl, 3,2 70of the dose, was found to be formed from 3,4-dihydroxyand to a minor extent from 4,4’-dihydroxy-biphenyl. During these experiments no evidence was found for a microbial dehydroxylation of 3,4,4,’-trihydroxybiphenylor 3,4-dihydroxybiphenyl. Thus, 3,4-dihydroxybiphenyl was not converted to 3-hydroxybiphenyl when administered to rats orally or by an intracaecal injection. By the same routes of administration 3,4,4’-trihydroxybiphenyl showed no sign of a p-de-

Table 4 . Urinary metabolites from biphenyl in rats with prevented bile flow. Urine collected daily for 4 days after oral administration of 25 mg (approximately 100 mglkg) biphenyl/ animal. Results are mean values as % dose, with numbers of animal in parenthesis. Urine (4-8)

+

Metabolite 1.day

2,day

Biphenyl 2-Hydroxybiphenyl 3-Hydrox ybiphen yl 4-Hydroxybiphenyl 2,5-Dihydroxybiphenyl 3-Hydroxy-4-methoxybiphenyl) 4-Hydroxy-3-methoxybiphenyl ) 3,4-Dihydroxybiphenyl 3,4’-l)ihydroxybiphenyl 4,4’-Di hydroxybiphenyl 3,4‘-Dihydroxy-4-methoxybiphenyl) 4,4’-Dihydroxy-3-methoxybiphenyl)

0.2 0.1 0.8 5.3 b)

0.1 0.1

0.5

3,4,4’-Trihydroxybiphenyl Total a) Trace metabolite b) N o t detected c) Combined quantitation

7.1 b)

3. 4. day

Total

0.1 0.2 0.3 3.4 b)

0.4 0.4 I .6 15.8

0.1

a)

0.1

0.2

0.4 0.6 4.5

0.3 0.5 4.2

0.3 0.5 2.8

1.o 1.6

11.5

0.2

0.3

0.2

0.7

0.5

0.9

0.6

2.0

12.7

14.0

8.5

35.2

PHENOLIC BIPHENYL METABOLITES IN RAT

427

hydroxylation reaction. An attempt to show this kind of reaction also failed when the compound was incubated with rat caecal microorganisms under anaerobic conditions. Thus, 3,4’-dihydroxybiphenyl appears to arise only from a second hydroxylation of a monohydroxylated biphenyl. Monomethylated derivatives of both 3,4-dihydroxy- and 3,4,4’-trihydroxybiphenyl were found in the rat urines and also when the respective catechols were incubated with the enzyme catechol-0-methyl transferase in vitro. The two monomethyl ethers of the former compound represent only 0.1 olo in the 96 hrs urines and the corresponding value of the latter is 0.9 Yo. Separation of the monomethylated derivatives was not achieved using packed columns but the capillary column was satisfactory (table 1). In this way it was found that the ratio of meta:para methylation of 3,4-dihydroxybiphenyl (1 mg) was approximately 0.5 in the COMT experiments and 2.5 when the catechol (100 mg/kg orally) was administered to rats. Similarly, the in vitro meta:para ratio when 3,4,4’-tnhydroxybiphenyl was used was approximately 0.5. Furthermore, some of this trihydroxy compound and the two methyl ethers were formed when 3,4-dihydroxybiphenyl was given. The meta:para ratio in this case was approximately 2.5. The effect of suppressing the metabolism of biphenyl and/or its metabolites by the intestinal microorganisms was studied in two rats given neomycin sulphate. The results are listed in table 3, from which it can be seen that the metabolic pattern was similar to that shown in normal rats. The role of the enterohepatic circulation of biphenyl metabolites was studied in five rats in which the bile flow was prevented. The results are listed in table 4, from which it can be seen that the main difference from the normal experiments was a doubling of the amount of urinary 4-hydroxybiphenyl. Minor differences in the amounts of some of the other metabolites also occurred. Faecal metabolites. The phenolic metabolites detected in the faeces 24 hrs after the rats were given biphenyl are shown in table 2. These metabolites were unconjugated and accounted for 4.7 Vo of the dose.

Biliary metabolites. The biliary metabolites from biphenyl in the rat are shown in table 5, from which it can be seen that 4-hydroxybiphenyl, 4,4’-dihydroxybiphenyl and 3,4,4’-trihydroxybiphenyl are the most prominent in the enzymehydrolysed bile. Considerable amounts of the mono-hydroxy compound are excreted after 6-7 hrs whereas excretion of the other two compounds is essentially complete at that time. The total amount of phenolic metabolites in the 24 hrs bile was 5.2 Vo of which 3.8 % was detected 6-7 after dosing,

428

T. MEYER AND R.R.SCHELINE

Discussion

Biphenyl is a fat soluble aromatic hydrocarbon which requires conversion into polar metabolites in order to be excreted from the animal body. The present study deals with the conversion of biphenyl into phenolic metabolites in the rat. It has substantiated the known qualitative metabolic picture in rats and has also identified some metabolites not previously reported. The proposed metabolic pathway of biphenyl in rats is shown in fig. 2. The total amount of phenols found in the rat urine 96 hrs after administration of biphenyl was 29.5 Yo, which is in contrast to the findings of 58 % by WEST et al. (1956), 2 2 % in 48 hrs by CREAVEN & PARKE(1966) and 41 96 in 24 hrs MEYERet al. (1976). The results reported by WEST et al. (1956) might reflect an induction of biphenyl-4-hydroxylation caused by the ingestion of the compound itself over a period of several weeks until 15 g was consumed. It is of interest to note that the higher value (41 % in 24 hrs) found by MEYERet al. (1976) using 14C-biphenylcompared with the value obtained in the present study, differs mainly because a free phenolic fraction was detected in the former investigation. This suggests the presence of phenols of unknown structure which are not amenable to gas chromatography under the conditions used, In the present experiments 4-hydroxy- and 4,4‘-dihydroxybiphenyl are the most prominent metabolites, and the latter is the main one. These findings are in contrast to earlier experiments in which 4-hydroxybiphenyl was re-

Fig. 2. Proposed metabolic pathways of biphenyl in the rat. The numbers refer to the compounds listed in table 1. Compound in brackets have not been detected. Major and

minor metabolic routes are indicated by thick and thin arrows, respectively. Broken arrows indicate possible but not verified metabolic conversions in the rat.

PHENOLIC BIPHENYL METABOLITES IN RAT

429

ported to be the major metabolite of biphenyl in the rat (WESTet al. 1956; CREAVEN & PARKE1966). A major point of interest in the present work is the detection of several previously unknown metabolites of biphenyl. The two monomethylated derivatives of 3,4-dihydroxybiphenyI have been reported to be formed from biphenyl in the rabbit (RAIG & AMMON1972) but their formation in the rat has not been described up to the present. The new metabolites found are 3,4’-dihydroxybiphenyl, 3,4,4’-trihydroxybiphenyl and its 3- and 4-0methyl ethers. The site of formation of 3,4‘-dihydroxybiphenyl was of interest because catecholic compounds are well known to undergo p-dehydroxylation as a result of the metabolic activities of intestinal micro-organisms (SCHELINE 1973). Thus, 3,4,4’-trihydroxybiphenyl which is found in the bile to an extent of approximately 1 % of the dose could be dehydroxylated to the 3,4’-dihydroxy compound. This does not appear to be the case, however, as no dehydroxylation was detected when the tri-hydroxy compound was incubated with rat caecal micro-organisms. 3,4’-Dihydroxybiphenyl was also found in the urine of biphenyl-treated rats in which biliary excretion had been prevented. The metabolite therefore appears to arise solely from 3hydroxy- and 4-hydroxy-biphenyl via a second hydroxylation reaction. The monomethylated catecholic derivatives were found both in vivo as urinary metabolites and in vitro in incubation studies with the enzyme catechol-0-methyl transferase. Because of the small amounts of the methyl ethers formed when biphenyl itself was given, the rats received 3,4-dihydroxybiphenyl in the experiments so as to determine the ratios of meta: para methoxy compounds. With both 3,4-dihydroxybiphenyI and 3,4,4’-trihydroxybiphenyl, the ratios found for the urinary methoxy metabolites were approximately 2.5. However, the ratios found in vitro were approximately 0.5. These findings are in accordance with earlier reports indicating that the meta:para ratios of 0-methylated catechols obtained in vivo are frequently much higher than those obtained in vitro (DALYet al. 1960; BAKKE1970). Biphenyl is preferentially hydroxylated at the 4-position and this appears to be a general and persistent tendency at any stage of the metabolic process. The only exception encountered is the conversion of 2-hydroxybiphenyl to its 2,4’-dihydroxy derivative, which is rather slorw in contrast to the formation of 2,5-dihydroxybiphenyI (MEYER,unpublished results). Furthermore ERNST(1965) found 21.4 9‘0 of the dose of 2-hydroxybiphenyl excreted as the 2,5-dihydroxy compound in the 48 hrs urine of rats. In the present investigation trace amounts of 2,5-dihydroxybiphenyl were occasionally found in the urine, as would be expected in view of the relatively small amounts of 2-hydroxybiphenyl produced. Variations in the amounts of the

430

T. MEYER A N D R. R. SCHELINE

latter metabolite observed in the present experiments may be related to age variations in the rats used since CREAVEN et ai. (1965) showed that it\ formation decreased with increasing age. The amount of phenols of biphenyl origin in the 24 hrs bile from rats was found to be 5.2 To of the dose, and the major metabolites detected were 4-hydroxy-, 4,4’-dihydroxy- and 3,4,4’-trihydroxy-biphenyl.In contrast, et al. (1967) and LEVINEet al. (1970) found only the two former MILLBURN metabolites in rat bile, but in a higher total yield (10-12 Yo). In the previously mentioned investigations, intraperitoneal doses of 3 1 and 100 mg/kg were used whereas the latter dose levels given orally were used in the present study. It is possible that variations in dose, dosage preparation or dosage route of administration may be involved in the differences found in biliary excretion. It is known, for example, that quantitative changes in the urinary metabolites of the related compound, trans-stilbene, result from changes in dosage of administration or route (SCHELINE 1974). The present results suggest that biliary metabolites are subsequently reabsorbed only to an insignificant degree. Both the pattern of metabolites

Table 5. Hiliary metabolites from biphenyl. Bile collected after oral administration of 25 mg (approximately 100 mg/kg) biphenyl/animal. Results are mean values as % dose, with numbers of animals in parenthesis. Bile Metabolite

6-7 hrs (2)

Biphenyl 2-Hydroxybiphenyl 3-Hydroxy biphenyl 4-H ydroxybiphenyl 2,5-Dihydroxybiphenyl 3-Hydroxy-4-methoxybiphenyl )

b) 0.1 0.5

1.5 b) 0.1

4-Hydroxy-3-mcthoxybiphenyl ) ‘) 3,4-Dihydroxybiphenyl 3,4’-Dihydroxybiphenyl 4,4'-Dihydrox ybiphenyl

0.1 0.3

1.9

3,4’-Uihydroxy-4-methoxybiphenyl) 4,4‘-Dihydroxy-3-methoxybiphenyl ) ‘) 3,4,4’-Trihydroxybiphenyl Total a ) Trace metabolite b ) Nut detected c ) Combined quantitation

24 hrs (3)

b) 0.7 3.8

5.2

PHENOLIC BIPHENYL METABOLITES IN RAT

43 I

and the amounts excreted in 24 hrs bile and faeces are similar (tables 2 & 5). Furthermore, the administration of neomycin sulphate which would be expected to reduce or prevent the bacterial hydrolysis of biliary conjugates in the intestine, did not result in appreciable changes in the extent or pattern of urinary biphenyl metabolites (table 3). The amount of phenolic metabolites excreted in the faeces after 24 hrs was not determined in the present study but it is not likely to be large as MEYERet al. (1976) found that only about 7 9'0 of the radioactivity was excreted in the faeces during a 96 hrs period following the administration of l4C-biphenyl. Moreover, the present results showing that about 5 olo of the dose is excreted in the faeces as phenolic metabolites, indicates that these compounds are the main types of biphenyl metabolites excreted by this route. Acknowledgements The authors are indebted to Norges Almenvitenskapelige Forskningsrhd and to Norsk Medisinaldepot for grants for the purchase of the GC/MS system and the gas chromatograph and integrator unit used in this study. We also thank Professor Dr. L. Horner, Institute of Organic Chemistry, University of Mainz, Germany, for his generous gift of a sample of 3,4dihydroxybiphenyl and Department of Chemistry, University of Bergen, for a sample of biphenyl. The technical assistance of Mrs. E. Tepstad, Mrs. A. Hetle and Mr. 0.E. Fjellbirkeland is greatly appreciated.

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The metabolism of biphenyl. II. Phenolic metabolites in the rat.

Acta pharmacol. et toxicol. 1976, 39, 419-432. From the Department of Pharmacology, University of Bergen, MFH-Bygget, 5016 Haukeland sykehus, Bergen,...
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