15.8. 1973 Specialia 953 this fraction showed a low mean delta value : E lcm/1% = strated ~3 that the plasma membrane of liver cells presents 0.55. enzymatic alterations at 14 days, this positive result must Discussion. The concept of a purely biologicaI anti- be considered an expression of a late alteration, unrelated oxidant activity of vitamin E, and an associated function to the causal mechanism of induction of the cellular of selenoamino acids as free radicals scavengers and injury. Furthermore, the existence of microscopic peroxide descomposers, has suggested lipid peroxidation necrotic changes in some of the livers in the prenecrotic in vivo as the original alteration in the pathogenesis of period cannot be excluded. Slight contamination of the dietary liver necrosis a,! Cellular membranes would be microsomal fractions with plasma membrane might damaged, since they are largely composed by polyun- account for the atypical curves observed in some exper- saturated fatty acids. iments. A large part of the argument in support of the lipid The present results stress the need for alternative peroxidation hypothesis derives either from experiments explanations. Mild lipoperoxidation damage comes too in vitro on the properties of antioxidants, or comparative late in the sequence of events leading to cellular necrosis studies with other experimental models, such as radiation to account for its pathogenesis. damage and ageing processes. A protective action of antioxidants compounds like DPPD, replacing the vitamin Zusammen/assung. Es wird festgestellt, dass der E in the diet, has been advocated as direct evidence for Einfluss yon Lipidperoxyden nicht ffir die Entstehung the lipid peroxidation mechanism 9. However, studies gewisser Formen der Lebernekrose verantwortlich ge- conducted by other authors failed to confirm the alterna- macht werden k6nnen. tive action of vitamin E and structurally different anti- oxidants as related to prevention of lipid peroxidation ~0, n E. A. MACHADO 14 and F, HAMILTON 16 It is interesting to note that in other non-toxic cellular necrosis, like renal necrosis in choline deficient rats, in Centro de Patologta Experimental, Facutlad de Medicina, which lipid peroxidation in vivo has been demonstrated, J.E. Uriburn 950, Universidad de Buenos Aires, Buenos DPPD leads to a decrease of the renal lesions while A ires (Argentina), and vitamin E fails to exert a similar protective action 12. The Research Institute o[ The Hospital [or Sick Children, Critical examinations of the lipid peroxidation hypothesis Toronto, (Ontario, Canada), 6 February 797& have concluded that the peroxide content in rat liver is not altered by the addition of vitamin E to the diet ~. The significance of peroxides detected by the widely used reaction of the thiobarbituric acid (TBA) with A. L. TAPPEL, Fedn Proc. 24, 73 (1955). malonaldehydes, has been seriously objected to as I~ K. SCHWARZ, in Liver Function (Ed. R. W. BRAUER; American evidence of the existence of lipid peroxidation in living Institute of Biological Sciences 1958) ; p. 387. tissues. It is presently believed that malonaldehyde is 11 j. BUNYAN, E. A. MURRELL, J. GREEN and A. T. DIt'LOCK, Br. J. metabolized in vivo through mitochondrial pathways, Nutr. 27,475 (1967). and therefore the TBA reaction would depend on perox- 12 A. J. MONSERRAT, A. K. GHOSHAL, W. S. HARTROFT and E. A. ides formed in vitro during the procedure6. PORTA, Am. J. Path. 55, 163 (1969). Ia E. A. MACnADO, E. A. PORTA, W. S. HAReROF% and F. HAMILTON, The results reported here, obtained by the method of Lab. Invest. 2,t, 13 (1971). detection of diene conjugates, indicate that there is no 1~ New address: The University of Tennessee Memorial Research evidence of lipid peroxidation during the different stages Center and Hospital, Knoxville (Tennessee 37920, USA). of the prenecrotic period, except for the plasma membrane ~5 Acknowledgments. The authors wish to thank Dr. A. K. GHOSHAL fraction at 21 days. Since it has been previously demon- for many valuable discussions.

Identification of Two New Metabolites of Gaffeine in the Rat Urine

During our recent studies i on the metabolism of - m* 155), 142 (169-HCN, m* 119.5) and 109 (194-CHsNCO ~H in the rat, we reported the isolation of the following and CO)] and its ditrimethylsilyl derivative II [peaks at metabolites from the chloroform-methanol (9:1) extract S re~e: 356 (M+) and 341 (M-CH~)3. Proton nuclear magnetic of the urine: (1.2%), theobromine (5.1%), resonance analysis (CDC1s solvent) indicated that in solu- paraxanthine (8.8%) and trace amounts of 1, 3, 7-trime- tion, metabolite A appears to be in equilibrium wittl its thyluric acid and 3-methyluric acid. In addition, two uni- open-chMn, N-formyl analog III. About 25% caffeine (iV) dentified metabolites, A (11.4%) and B (1.3%), were iso- was also found to be present in the solution (Scheme I). lated. The present communication deals with the struc- Oxidation at the 8 position of the purina ring to yield 8- ture elucidation of these 2 new metabolites of caffeine. hydroxy derivatives has been previously observed in the The thin-layer chromatographic (TLC) and spectral (IR, rat with guanine-3-oxide 4 and purine s itself. UV and mass) characteristics of the isolated metabolites A and B were found to be markedly different from those of the known mono-, di-, and trimethyl derivatives of xan- thine and uric acid 1, 3. The major metabolite A appeared 1 K. L. KHANNA, G. S. RAO and H. H. CORNISh,Toxic. appl. Pharmac. to be a polar compound. It readily dehydrated to caffeine 23, 720 (1972). under TLC and gas chromatographymass spectrometric The chldroform-methanoi (9:1) extract of the urine accounted for (GC column: 1%-OV-17, temperature 190~ conditions about 35% of the ingested radioactive caffeine out of which about and as such it is difficult to isolate this metabolite in pure 9% was found to be unchanged caffeine. G. S. RAO, K. L. ]

Scheme I. b4.0 6 3.05 h 3,2 0 CH 3 ~3.6 JL ~, O CH~ O CH3 CH~--,N" ,"J"\ 63.5CH_,N- /~ I~X.~ .OR _._ ~,3.~ ..1...N--O--H[ 6Z98

o O O ~ "~N~NH2 CH3 I 63.4 CH~ OH 3 6 535 53.35 IV 1-25% ) l Ig (~50%) (~25%) I. R=-H II. R=TMS

Structure V (3, 6, 8-trimethylallantoin) may be assigned was recovered from the chloroform-methanol (9 : 1) extract to the minor metabolite ]3 based on its mass spectral frag- of the urine. The 3 mono-N-dealkylated products, theo- mentation pattern [peaks at m/e: 200 (M+), 143 bromine, paraxanthine and theophylline, isolated from the (M-CH~NCO, m* 102 and 115 (M-CO)~. The metabolite chloroform-methanol extract account for about 15% of B gave positive reaction to the YOUNG-CoNwAY test 6 the ingested radioactive caffeine. Hydroxylation followed which confirmed that it is a derivative of allantoin. Allan- by reduction of caffeine apparently leads to the isolated toin is known to be the major metabolite of purine, xan- major metabolite, 1, 3, 7-trimethyldihydrouric acid (I). thine, and hypoxanthine in the rat s, ~. FRANKE and HAHN 8 Dehydrogenation of I gives 1, 3, 7-trimethyluric acid have reported earlier that certain microorganisms meta- which then undergoes N-dealkylations to form 3-methyl- bolize methylxanthines, such as caffeine, to methylallan- uric acid. However, the more favored mode of degradation toin. DRYHURST and I-IA~sE~ 0 have recently identified 3, 6, 8-trimethyl allantoin (V) as a product of electrochemical oxidation of caffeine. Photochemical oxidation of methyl- xanthines is also known to lead to methylallantoins ~0. 6 E. G. YOUNG and C. F. Co~wA% J. biol Chem. 1#2, 839 (1942). Sequential base and acid hydrolyses of allantoin and its N-methyl Scheme II describes the proposed biotransformations derivatives quantitatively yield glyoxylic acid which can then be of caffeine in the rat. About 9 ~o of the unchanged caffeine readily detected by the development of the characteristic chromo- phore, ]~max. 515 nm under the Rimini-Schryverreaction conditions. 7 H. G~TLER, P. M. ROLL, J. F. TI~KER and G. B. BRowN, J. biol." 0, CM~ Chem. 178, 259 (1949). s W. FRANKE and G. E. HAHn, Hoppe-Seyler's Z. physiol. Chem. 301, 90 (1955). NH--C 9 G. DRYHURSTand B. H. HANSON,J. electroanalyt. Chem. 30, 407 I II I H [1971). GH 3 O GH3 10 I. R. POLITZER, G. W. GmFFIN and J. L. LASETER, Chem. biol. g Interactions 3, 73 (1971).

Scheme II. Hydroxylation] Reduction Caffeine 3- 1, 3, 7-Trimethyldihydrouric acid (11.4%) (Unchanged, 9%) (I)

I N-Demethylations I Dehydrogenation N-Demethylations 1,3,7-Trimethyluric acid (Trace) 3-Methyluric acid (Trace) Theobromine (5.1%) Paraxanthine (8.8%) I Oxidation Theophylline (1.2) % 3, 6, 8-Thimethylallantoin (1.3%) (V)

Oxidation + 1,6, 8-Trimethylallantoic acid

Hydrolysis + Giyoxylic acid Methylurea 1, 3-Dimethylurea 15.8. 1973 Specialia 955 of trimethyluric acid appears to be the oxidative ring Of experimental animals represent only a fraction of the opening of the pyrimidine ring to form 3, 6, 8-trimethyl- urinary metabolites of caffeine~-~a Our results seem to allantoin (V) which can then be further oxidized and explain the observed difficulty in recovering caffeine meta- hydrolized to more polar products such as 1, 6, 8-trimethyl- bolites from the urine since methylated allantoins, allan- allantoic acid, glyoxylie acid, and mono-, and dimethyl- toic acids and derivatives are highly polar and not . easily extractable. Also these polar compounds may con- The hitherto known metabilites of caffeine viz, methyl- stitute the unidentified metabolites encountered by earlier xanthines and methyluric acids, isolated from the urine investigators ~.

~1 H. H. CORNISH and A. A. CHRISTMAN, J, biol. Chem. 228, 315 (1957). Rdsumd. L'acide 1, 3, 7-trim6thyldihydro-urique et la ~2 T. OroMo, Nippon Univ. J. Med. 18, 77 (1959); through Chem. 3, 6, 8-trim6thylallantoine out 6t6 identifi6s comme m6ta- Abstr. 61, 9914 g (1964). bolites nouveaux de la caff6ine dans l'urine de rat. 13 A. W. BURG and M. E. STEIN, Biochem. Pharmae. 21, 909 (1972). 14 This investigation was supported in part by U.S. Public Health G. S. RAO ~5, If. L. KHANNA and H. H. COI~NISH Service Grant No. GSi 15269. The authors are grateful to Dr. It. M. FAL~S for many helpful discussions. ~s Present address: Laboratory of Chemical Pharmacology, National Department of Environmental and Industrial Health, Heart and Lung Institute, National Institutes of Health, Bethesda School o/ Public Health, The University o/ Michigan, (Maryland 20014, USA). Ann Arbor (Michigan 48104, USA), 12 March 7973.

Effect of Medium pH on p-Aminohippurate Accumulation by Slices of Rat Renal Cortex ~

Several techniques have been employed to study the slices from this species. For comparative purposes the transport of organic ions by the kidney, including effect of pH on PAH accumulation in rabbit and dog clearance2, 3, micropuncture and microperfusion 4,~ and tissue was also determined. isolated tubules~,L Probably the most widely used Methods. Adult mongrel dogs, New Zealand rabbits and technique is the in vitro slice method of CRoss and Sprague-Dawley rats were used in these studies. Dogs were TAaGARTS. Even though the method is routine in many anesthetized with pentobarbital sodium (30 mg/kg, i.v.) ; laboratories optimal conditions for incubation have not rabbits and rats were stunned by a blow on the head been completely elucidated. For instance, CRoss and TAG- Kidneys were quickly removed, weighed and placed in GART s observed that accumulation of the organic acid ice-cold saline. Renal cortical slices were prepared free p-aminohippurate (PAH) by rabbit kidney cortical slices hand and briefly kept in cold saline until incubated. Slices was dependent upon temperature and oxygen but was (approximately 100 rag) were incubated for 90 min using independent of pH over a relatively narrow range either 0.1 M sodium phosphate buffer or 0.015 M 2- (pH 7.0-7.8). However, COPENgAVER and Davis 9 found amino-2-methyl-1, 3 propanediol (propanediol) buffer, in that over a wider pH range (5.0-12.0) the greatest medium containing 7.4 • 10 -5 M PAH. All incubations accumulation of PAH by rabbit renal cortical slices was were carried out in duplicate in a Dubnoff apparatus at at pH 8.1 to 8.3. In contrast, Ross et al. ~0 found the grea- 25 ~ under a gas phase of 100% oxygen. test accumulation of PAH by dog renal cortical slices to The pH of the incubation medium was initially adj usted be at pH 7.4. To our knowledge comparable studies have to values ranging from 6.0 to 9.0. Renal cortical slices not been conducted in tissue from the rat. Inasmuch as from individual dogs or rabbits were incubated in medium rat tissue is extensively employed for studies of renal at several different initial pit values in each experiment. transport in this laboratory, it was of interest to determine Kidney slices from 4 rats were pooled and treated as the effect of pH upon PAH accumulation by renal cortical tissue from one animal. In the experiments with dog tissue phosphate buffer was used throughout the pH range, while propanediol was used in all rat and rabbit studies. Unless stated otherwise, the reported pH values : : Dog are those measured after incubation. The final pH range 12 ~--.. --.-e Rat '\ was from 6.4 to 8.4. The effect of medium pH on the rate o ...... Rabbit / ' of PAH uptake was determined in phosphate buffer at initial pH values of 7.4, 8.0 and 8.5. Slices of rat renal ....~ ' ".... cortex were incubated for periods of time ranging from 5 to 30 rain. 212

1 Supported in part by USPHS grant No. AM-10913. G.H. MUDC-Eand J. V. TAGC-ART, Am. J. Physiol. 161, 191 (1950). /2 /4 7.'6 /8 8'.0 8'.2 8'.4 a G. A. TANNER and M. T. ISENBERG, Am. J. Physiol. 219, 889 (1970). Final pH 4 G. A. TANNER, Am. J. Physiol. 212, 1341 (1967). P. DEETJEN and H. SONNENBERO, Arch. ges. Physiol. 285, 35 (1965). Effect of medium pH on PAH accumulation (S/M). pH values shown 6 M. B. BURG and J. ORLOFF, Am. J. Physiol. 203, 327 (1962). are those at the end of 90 min incubation. Eaeh point represents the K. C. HUANO and D. S. T. LIN, Am. J. Physiol. 208, 391 (1965). mean (~ SE) obtained in 5 duplicated experiments. When no verti- s R. J. CROss and J. V. TA~GART, Am. J. Physiol. 161, 181 (1950). cal line is shown the variation is within the radius of the circle. In in- J. H. COPE~'HAVERand J. R. DAVIS, Proc. Soc. exp. Biol. Med. 7lP, dividual experiments a single dog or rabbit was used. For rat experi- 611 (1965). ments, the kidneys of several animals were pooled and then treated as s0 C. R. Ross, N. I. P~ssAH and A. FARAn, J. Pharmac. exp. Ther. 160, one. 381 (1968).