NIGluessidation of amobarbital in the eat1

GIOVANNA CARRQ-CIAMPI,' M A L L I ~ JURIMA, DEZSO KABAR, BING K . TANG, AND WERNER KALOW Departrnenf of Pharmacology, University of Toro~ito, Toro~~to, Ont., Canada M5S ZAH

Received November 1 , 1984

CARRO-CIAMPI, G., M. JWRIMA. D. KADAR. B. K. TANG, and W. KALOW. 1985. N-Glucosidatic~n of amobarbital in the cat. Can. J . Phy siol . Phamacol . 63: 1263 - 1266,

N-Glucasidation is a novel pathway of barbiturate metabolism, so far known to occur only in man. A search for an animal modei, conducted through in vitro screening, revealed that amobarbital-N-glucoside was formed in liver preparations from the cat. The presence of arnobubital-N-glucoside was demonstrated in cat urine, foIlowing i.p. administration of amobarbital.

CARRO-CIAMPI, G., ha. SURIMA, D. KADAR, B. K . TANG et W. KALBW. 1985. N-Glucosidation of amobarbital in the cat. Can. J. Physiol. P h m a c s l . 63: 1263- 1266.

La N-glucosidation est une nouvelle voie du mktabolisme des barbituriques que l'on avait toujours cm exclusive B I'humain. La recherche d'un modkle animal, effectute par une Ctude in vitro, rCvela la formation d'amobarbital-N-glucoside dans des prkparations de foie de chat. On a BkmontrC la prksence d'amobarbital-N-glucoside dans I'urine de chat suite A I'adrninistration imp. d'arnobarbital.

[Traduit par le journal]

Introduction Barbiturate N-glucosides are major metabolites of the barbi-

turates in man, and were identified for the first time in human urine (Tang et ai. 1948, 1949). Only two other reports of N-glucosidation in mammals have appeared so far (Duggan et al. 1944; Paulson et al. 1981). The discovery of barbiturate N-glucssides prompted a search for an animal species capable to effect barbiturate N-glucosidation. The availability of such an animal model would be useful in further studies of the newly discovered metabolic pathway. In vivo studies conducted in dogs, mice, hamsters, guinea pigs, and albino rats failed to reveal any amobarbitril N-glucoside in urine, except trace amounts in albino rats (Tang et al. 1980).

An in vitm study with human liver preparations showed that amobarbital N-glucoside could be detected and that its for- mation occurred in microsomes and was UDP-glucose de- pendent (Tang and Carro-Ciampi 1980). Application of the above method of study, simplified to the use of crude tissue homogenates, appeared to offer a convenient approach to the search for an animal model. We report here on an in vitro screening which identified the cat as a possible animal model of barbiturate N-glucosidation. An in vivo experiment was then conducted to test whether amobarbital-N-glucoside was formed in the cat after i.p. administration of amobarbital.

Methods In vitro screening

Human specimens, i.e., liver (n = l ) , kidney (n = 2), lung (n =.

2), and large intestine (n = I ) , were obtained from surgery cases at the Toronto General Hospital. They were quick frozen in liquid nitro- gen, stored at -80°C and tested within I month. An additional human liver specimen was obtained from a kidney transplant case at the Toronto General Hospital and was tested after periods of -80°C storage for 9 and 29 months. Single specimens of monkey. cow, pig, duck, rabbit, cat, dog, and albino rat livers had k e n obtained at different times and from various sources. Duration of storage at -80°C was not longer than 24 months except in one case (cat No. 1 liver. stored for 36 months). Following the initial screening, cat livers were obtained from three additional animals (cats Nos. 2, 3, and 4), stored at -80°C, and tested within 4 months.

"his work was supported by a grant from the Medical Research Council of Canada.

'Author to whom correspondence should be addressed.

A11 nonradiolabelled chemicals were purchased from Sigma Chem- icals (St. Louis, MO). ['4C]amobarbital (['"C]Amo), with a specific activity s f 5.C) Ci/mol (1 Ci - 37 GBq), and 3'-hydroxyamobarbital (C-OH) were synthesized as reported earlier (Tang et ctl. 1975). Amo- barbital N-glucoside (N-GIu). to act as a standard, was prepared as described earlier (Tang and Carro-Ciampi 1980). [ncubations with I mL of tissue homogenate, prepared by homogenizing 0.5 - I .O g of tissue in ice-cold I . 15cZ~ KCB. were carried out for B h at 37°C. Except otherwise stated, incubation mixtures contained the following in a 5-mL total volume: 25 pM ["C]Amo, 76 nmM KH2P0, (pH 7.4), 92 mM KCl, 5 mM UDP-glucose. 2 mM MgGl2, 0.4 mM NADP, 1 mM NADH, 4 ~ I M glucose-6-phosphate and 2 U glucose-6- phosphate dehydrogenase.

Ninety-nine percent extraction of radioactivity from incubation mixtures, to which 0.2 mL of 1 M HGI and saturating (NH4)2SO4 were added. was achieved with 3 X 5 mL volumes of ethyl acetate. The analysis of extracts was carried out by a two-dimensional thin-layer chromatographic (2 X D TLC) method developed previously (Tang and Carro-Ciampi 1980). It involved a first chromatography, using solvent system A (n-butyl chloride : dioxane. I : l), to 15 cm, and a second chromatography at a right angle to the first, using solvent system B (n-butanol : water, 93 : 73, to I2 cm. The merit of the methud was to permit separation of N-Glu from materials of lesser as well as greater polarity. Metabolite formation was expressed as 1-h yields, in picomoles per gram of tissue. Minimum detectable I-h yields were 50 pmol/g tissue.

In vivo test Five nonconditioned cats, two male, three female. wcighiang from

3.8 to 4.4) kg, obtained through the Division of Laboratory Animal Science, Faculty of Medicine, University of Toronto, wcrc housed in the animal quarters, one per metabolism cage. They had food and water cad Iihitum throughout the study.

The source of chemicals was as above. Injectable solutions obtained by mixing appropriate amounts of freshly prepared cold and radio- labelled amobarbital solutions were filtered through Millex@ Filter Unit, type HA, 0.45 pm (obtained from Millipore Laboratory Prod- ucts, Mississauga, Ont. ,) immediately prior to use. Each cat received, i.p., 20 mg/kg and 3.5 pCi/kg of C1"C]Amo. Total urine and feces were collected daily and frozen. Urine was stored at -20°C until analysis. Feces were freeze-dried, pulverized, and stored in a des- sicator at room temperature. Wash soiutions. obtained daily by rinsing the funnel base of metabolism cages with water, were collected and stored frozen.

Radioactivity was counted as previously described (Tang et al. 1975). Powdered feces (approximately 50 mg) were burned in the combustion apparatus of Kalberer and Rutschmann ( 1 94 1) and the released '4C02 was trapped in 15 snL of ethanolamine in methanol

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1264 CAN. 9. PHYSIOL. PPBARMACOI>. VOL. 63, 8985

( 15 : 85, v/v). To 1 mL of this sojution was added 10 mk of butyl-PBD (Sigma Chemicals, St. Louis, MO) in toluene (0.73: 100, w/v). Radioactivity counting in nonaqueous liquid samples was carried out by adding to sample aliquots (100-200 pL) 2 mL methanol and 5 mL of the butyl BBD - toluene mixture described above. The radio- activity in urine and other aqueous saniples was counted by adding to sample aIiquots (100-200 FL) I mL water and 15 mL Aquasol (New England Nuclear, Boston, MA). Consistency between the different methods of radioactivity counting was monitored by obtaining differ- ent measurements on standard samples. Correction factors were ap- plied when necessary~

Aliquots of urine ( 1 -2 mL) were extrdcted with three 5-mL por- tions of ethyl acetate in the presence of saturating (NH4)2S04. Extrac- tion efficiency was the radioactivity in the extract as a percentage of that in the aliquot sampled. The effect of incubating urine with GBusulase'?" (Sigma Cltemicals, St. Louis, MO) (10 000 U Glusulase/rnL urine, pH 4.5, 37°C for 16 h) was determined. A sequential TI,@ (solvent A, then solvent B along the same dimension) was performed on whole urine, purified by passage through XAD-4 resin (BDH, Toronto, Ont.) as described earlier (Tang pt ul. 1979). Two samples of cat urine were analyzed according to tlac gas chro- matography - mass spectrometry (GC/MS)I method previously devel- oped (Tang ct ul. 1977). Analysis of all the urine samples collected daily was carried out by subjecting ethyl acetate extracts of sample aliquots to 2 X D TLC, according to the method described above (Tang and Cam-Ciampi 1980). The number of radioactive counts associated with a TI,C peak, expressed as a percentage of total counts on the TEC plate, was corrected for the extraction efficiency. The resulting value was converted to percent of the administered dose, based on the radioactivity present in the entire daily urinary sample. Minimurn detectable levels, corresponding to TLC peak radioactivity twice that of background. were calculated to be equfvaIent to 0.05% of the administered dose. Daily urinary excretion data were summed to give amounts recovered in 6 days of urine collection.

Results In vitro srr~eizing

Human liver specimens from surgery and frona kidney trans- plant did not differ in C-OH or N-Glu yields (Table 1). N-Glu yields by human colon. kidney, and lung were lower than the limit of detectability (data not shown), although in the two latter organs 6-OH fomation did occur (200-5OU pmol/g tissue). A human liver specimen tested after 29 months of -80°C storage produced N-Glu yields comparable to those observed upon earlier testing, 20 months previously (Table 1).

Animal liver specimens gave rise to 6-OH yields which were equal to or greater than those of human liver, except in one case (3080 pmol/g liver in the dog specimen). N-GBu fomation in dog, cow, duck, pig, rabbit, and monkey liver was not de- tectable (data not shown); it was barely detectable in rat liver (60 pmol/g liver). Iiacubations with cat liver homogenates yielded a product with chromatographic behaviour identical to that of synthetic N-Glu, along two solvent systems (radio- chromatograms not shown). No interfering peaks were ob- served on either solvent system TLC. Cat liver specimens. tested after periods of -$PC storage ranging from 4 to 36 months, gave rise to N-Glu formation in amounts several times the limit of detectability. Amobarbital N-glucosidation was independent of the NADBH-generating system but dependent on UDB-glucose and MgC12 (Table 1).

%aim 1 . Amobarbital-N-glucosidc formation in livera

Species N-GBu C-OH

Cat (No.) I 170 80 000 2 350 7 150 2" 570 ND 3d 420 ND 4d 4-40 ND

- - - - - - - -

NOTE. N-Glu, amobarb1tal-N-gBu4.oslde, C-ON. 3'-hg dmxg - amobarbital; ND. not de tem~ned

"Cofactor concentrations and incubation conditions are de- scribed in Methods. Yields are in pisornoies per gram of liver.

"Liver specimen obtained from kidney transplant (K43 or surgery ( S 1 ).

'Time of storage at -80°C was 29 months. *Cofactors for the NADBH-generating systern were omitted.

No. 5 , so the data were not included. Jn all other cases, con- tamination of feces by urine was likely to have been minor since 5% or less of the administered radioactivity was recov- ered in cage washings (Table 2). The feces : urine radioactivity ratio varled somewhat in different cats (Table 2).

Ethyl acetate extraction efficiency of cat urine was 80 - 85%. It was not changed after Glusulase" incubation, showing that the unextracted materials were not glucuronides or sul- fates, The lack of any effect of Glusulase@ incubation on the percentage of polar materials was confirmed on radio- chromatograms obtained by sequential TLC of XAD-purified whole urine. Analysis of cat urine ethyl acetate extracts by 2 x D TLC showed the presence of a peak with R f identical to that of synthetic N-Glu on both solvent systems A and B. Owing to the two-dimensional combination of solvent systems with dif- ferent polarities, the M-Glu pea&, although relatively srnall in size, could be visualized as distinct from peaks which, on either one of the solvent system TLC alone, would have produced interferences (radiochromatograms not shown). With the ex- ception of three samples (one from day 5 and two from day 61, N-Glu was present in amounts above the minimum detectable in samples collected from all five cats. Urine samples analyzed according to the method previously described (Tang et a!. 1977) showed the characteristic spectra of 6-QH and N-Glu (GC-MS tracings not shown). Table 2 shows the cumulated excretion of ['4C]Amo and metabolites M-GIu and C-OH, during 6 days of urine collection. N-Glu represented the minor excretory product whereas the major urinary product was C-OH. Other materials, not yet identified, representing from 3.0 to 5.5% of the administered dose, were recovered in the ethyl acetate extracts. The remainder of the urinary radio- activity was composed of unextractable materials. As apparent in Table 2, the N-Glu : C-OH in urine varied somewhat from cat to cat but was, on average, approximately 1 :60. The N- Glu : C-OH ratio in cat No. 5 urine was 1 :44.

In vivo test Discussion On average, 72% of the radioactivity administered was re- An in vitro screening was conducted for smobarbital N-

covered in urines and 116% in feces, during 6 days of collectiow glucosidation, which had been demonstrated previcusly in (Table 2). These average figures were derived from informa- human liver (Tang and Carro-Ciampi 1980). The screening was tion on four cats only: spillage of urine occurred with cat carried out on liver preparations since samples of human colon,

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TAI3r.e 2. Radicbaetivity recovered folIowing ['"Jarnobarbitid administration

Urine Cage

Species washings Feces 'Fotal Amo A'-Glu C-OH

Cat" (No.) 9' 10.5 18.9 38.1 1.15 0.71 31.6 6 5.2 19.8 68.1 2.50 1.55 50.3 7 B .8 6.0 86.0 1.35 0.42 65.8 8 I .7 16.0 60.5 3.61 0.73 40.8 9 1.4 20.4 73.8 3.06 0.78 51.8

Man' (No.) I 5 79 0.7 23 47 2 4 92 0.8 17 5 1

NOTE: Amo, arnobasbital; N-Glu, amobarbital-N-glucoside; C-Ot?, 3'-hydroxy - amoharbital.

"Figures represent radioactivity recovemi &tiring 6 days, as percent of the ati~iir~istered tinse of anlnRarbita1 (20 rng/kg, i .p.) .

"n unestirnated amount of' urine was accidentally lost: data fiom this animal wcre not includd ir~ average figures reported in ttme text.

cFigures were from data previcinsly puhllshed (Tang cl cii. 1975: 'Farip t.1 01. 198U): they represent radioactivity recovered durinp 5 days, as percent of the ingested dose of arnrpbarbltal ( 1 20 mg).

kidney, and lung failed to provide evidence of N-glucosidation. Incubations of liver honmogenates and ['4C]Amo contained UDP-glucose and MgC12 which are cofactors for N-gluco- sidation, as well as the NADPH-generating system, necessary for oxidative metabolisna. Measurennents of the latter were carried out as a general indication of tissue activity: we could thus exclude the possibility of artefact in any of the liver prep- arations used. Storage sf human (K6) and cat (cat No. 1) liver samples for 29 and 36 months, respectively, did not result in lack of N-glucosidation (Table 1). The presence of barely de- tectable N-Gtu yields in albino rat liver arid the absence of detectable N-Glu yields in dog, duck, cow, pig, rabbit, and monkey liver, therefore suggested that arnobarbital N- glucosidation in these species was either absent cpr very low. OUT in vitro findings agreed with observations in the dog and albino rat, previously investigated if^ vivo (Tang el uk. 1980). Detectable N-GIu yields occurred in cat Bivcr, provided UDP- glucose and MgGHZ were present (Table B ). The Iatter require- ment had also been observed in the case of human liver (Tang and Gano-Ciaanpi % 980), and added further evidence that the metabolite was a conj~agate of glucose. These in vitro results, therefore, pointed to the eat as a possible animal lamodel of barbiturate N-glucosidation.

To substantiate these results by in vivo evidence, an experi- ment was conducted on five cats which received i.p. adminis- tration of 20 mg/&g ['"]Anno. All daily urine sarraples collected for 6 days were analyzed, by extraction with ethyl acetate followed by 2 x D TLC of the extracts. In the urine samples ccallected. a product with chromatographic behaviour identical to that of synthetic N-Gler was found to be present. Analysis by GC-MS provided confirmation of these results. We therefore concluded that the cat is capable of amoharbital N-glucosidation, a novel metabolic pathway (Tang et al. 1978, 1979), which so far is not known to C)CCBI~ in species other than man (Tang et a/. 1980). It is noteworthy that the cat should form a glucosyl conjugate, since this species was found to be low in drug gl~lc~~ronyl-con.iugation capacity (Williams 19.78). Future experiments should investigate whether the cat is able to conjugate the other substrates which have been reported to undergo N-gBucosidation in rnarnmals, namely: phenobarbital

(Tang st al. 19791, sulfamethazine (Paamlson et a!. 1'381), and certain triazole derivatives (Duggan rt ak. 1944).

A detailed comparison of the overall fate of arnobarbital in cat and man was not an intended aim of this study. However, some interesting observations dcserve a brief comment. In the in va'vcs test on cats, N-Glu was found to be a nainor urinary excretion product of atnobarbital, and was approximately 60 times lower in amount than C-OH. In most laenman kolunteers investigated previously, N-GBU was a major urinary com- ponent, and represented an amount equal to or half that of C-OH (Kalow et aka 1978). Data from two huinan volunteers were obtained from previous studies (Tang ct cal. 1975; Tang e t ak. 1980) and are presented in Table 2. In vitl-ha, incubations of arnobarbital with liver ho~a~ogenates produced N-Glu and C-OH yields which did not differ widely between cat and man (Table 1). It was puzzling to find that, based on in ~.irro data, the arnobarbital N-glucosidation capacity of cats was roughly conlparable to that of man, whereas it appeared to be much lower than that of man, based on in vivo urinary data. On thc other hand, 16% of the ['4C]Arno ada~niniskesed i .p. to cats was recovered in feces during 6 days of collection (Table 2). Al- though the fecal : urinary ratio varied somewhat from cat to cat, fecal excretion was observed in all the animals and indi- cated that biliaq excretion occurred. A procedure for quan- tifying ['"]Amo and metabolites in feces was not available fronm previous studies and will have to be worked out sepa- rately, due to diff3culties inherent to the nature of fecal materials. Therefore, we do not know at the present time whether N-Clu is a component of the radioactivity excreted in cat feces. However, the discrepancy in estimates of the N- glucosidation capacity sf cats apparent between fra vivrs urinary data and in vitrn data would be explained if biliary excretion of N-GBu occurred. In two human volunteers, studied previously, the radioactivity recovered in 5 days of feces collection fol- lowing oral administration of [''@]/Arne was found to have been 5% or less (Table 2), but the composition of feces had not been examined (Tang et al. 1975). Subsequent studies, based on Barge numbers of human volunteers, showed by indirect evi- dencc that in the majority of sub.jects fecal excretioia was likely to have been minor (Kalow et ak. 1978). However. exceptional subjects were encountered who, following ingestion of amo- barbital, excreted little or no N-Glu in urine (Kalow st a/. 1978). Whether, in these subjects. N-Glu was synthesized and excreted via the biliary route or was not synthesized, remains to be ascertained. In conclusion, we would like to suggest that future investigations of the fate of amobarbital in the cat should include biliary excretion studies. These will serve to assess further the newly discovered animal mode1 of anlobarbital N- glucosidation, and, possibly, may help to explain findings en- countered in man.

Acknowledgements We acknowledge the excellent contributions of Ms. R.

Tyndale, Mr. T. Fecycz, and Mrs. IS. Yilrnaz.

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