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Identification of Two New Metabolites of Caffeine in the Rat Urine

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Identification of Two New Metabolites of Caffeine in the Rat Urine 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 caffeine- 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: theophylline (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. ]<HANNA and H. H. CORNISH, J. pharm. Sei. form. We have assigned structure I (1, 3, 7-trimethyldihy- 61, 1822 (1972). drourie acid) to the metabolite A, primarily on the basis of 4 G. ST6HRER and G. t3. BROWN,J. biol. Chem. 2d4, 2494 (1969). the mass spectra of the metabolite [peaks at re~e: 212 .5 M. P. GORDON, O. x-~. INTERI~RIand G. t3. BROWN, J. biol. Chem. (M+), 194 (M-H~O), 184 (M-CO, m* 159.5), 169 (184-CIta, 229, 641 (1957). 954 Speeialia EXPERIENTIA 29/8 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 urea derivatives are highly polar and not ureas.
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