ABSTRACT. One experiment was conducted in which radioactively labeled purine bases (adenine, guanine, hypoxanthine and xanthine) were individually.
Metabolism of Orally and Intravenously Administered Purines in Rats1 D. A. SAVAIANO, C. Y. HO, V. CHU ANDA. J. CLIFFORD2
Department of Nutrition, University of California, Davis, CA 95616
Dietary purines, which include adenine, guanine, hypoxanthine and xanthine are extensively absorbed and as much as 7% of the radioactivity from a single oral dose is recovered from the tissues of rats after 24 hours (1). The retention of dietary adenine by body tissues is significantly greater than that of guanine, hypoxanthine or xanthine (1, 2). Furthermore, the retention of a single oral dose of adenine by body tissues is much less when given to fed rats compared with fasted rats (2).
acid in urine per unit amount of purine given. Although the catabolism of certain purine bases and nucleotides by gut sacs has been examined in vitro by several workers, the present report is the first to examine in vivo the possible influence of the gut on the fate of exogenous purines. Wilson and Wilson (4) found significant catabolism of adenosine monophosphate
An earlier individual
'Supported by USPHS AM16726 and Agricultural Experiment Station H28SO^The authors thank R. J. CraneR. E Shrader, T. A. Kunisaki, and C. K. Clifford for skilled technical assistance.
humans
Study (3) has purines when , , , rr
yield
also shown that given Orally tO f
dînèrentamounts
ot uric
Received forpublication 18February1980.
1793
!TOwhomreprintrequestsshouldbesent.
Downloaded from jn.nutrition.org by guest on July 14, 2011
ABSTRACT One experiment was conducted in which radioactively labeled purine bases (adenine, guanine, hypoxanthine and xanthine) were individually given intravenously to young adult rats and the recovery of radioactivity in urine and gut, gut content and liver was measured at the end of the next 24 hours. The total recovery of radioactivity from orally and intravenously administered adenine was measured in experiment 2. A third experiment measured the recoveries of radioactivity from oral and intravenous adenine in a wider variety of tissues and organs than in experiment 1. The chemical identities of the urinary end products of the metabolism of orally and intravenously administered adenine were compared in a fourth experiment. When purines were given intravenously, significantly more of the administered radioactivity was recovered in urine from rats given guanine, hypoxanthine or xanthine compared with those given adenine. The greater recoveries of radioactivity in urine were associated with smaller recoveries in tissues. A larger proportion of intravenously compared to orally administered radioactivity from adenine was incorporated into all body tissues, and this was most pronounced in glandular and lymphoid tissues. The primary urinary end product of both orally and intravenously administered adenine was allantoin. The absorption of individual purines from isolated rat gut sacs was evaluated in a fifth experiment. A significant proportion of unaltered adenine crossed the mucosal to serosal barrier of intestinal sacs whereas unaltered guanine, hypoxanthine or xanthine did not cross into the serosal fluid. These results show that the intestinal metabolism of dietary adenine is uniquely dif ferent from that of guanine, hypoxanthine or xanthine. J. Nutr. 110: 17931804, 1980. INDEXING KEY WORDS purines •oral •intravenous •gut sacs
1794
SAVAIANO ET AL.
MATERIALS AND METHODS
Five experiments were performed. In all experiments, male Sprague-Dawley rats (Simonsen Laboratory, Gilroy, CA) which were individually caged and ac climated to a commercial closed formula rat diet (Rat Chow, Ralston Purina, St. Louis, MO) for several days were fasted overnight. On the day of the experiment, rats were either fed (referred to as fed rats) or not fed (referred to as fasted rats) a single meal of 2.0 g of a modified
purine-free diet (2) at 0800 hours. The modifications of the purine-free diet (8) included replacement of the tapioca starch, the Hawk-Oser mineral mix and the safflower oil with glucose, the Zeman et al. (9) mineral mixture and olive oil, respectively. Intravenous injections. Twenty fed and twenty fasted rats (100 ±16 g) were used in the first experiment. One hour after meal feeding, i.e. 0900 hours, 8 /¿Ciof [8-14C]adenine (specific activity 56 Ci/ mole: all purines from ICN Pharma ceuticals, Irvine, CA) in 0.2 ml of 0.15 M NaCl were injected via the tail vein into five fed and five fasted rats. In addition, 8 fid of [8-14C]guanine, hypoxanthine and xanthine (specific activities 57, 55 and 54 Ci/mole, respectively) were each given separately to five fed and five fasted rats in a manner identical to the injection of adenine. The 40 rats were placed in individual metabolic cages and urine and feces were collected during the next 24 hours. At the end of this period, the rats were killed by decapitation. The entire gastrointestinal tract was re moved and the contents separated by flushing with warm water. The liver was also removed. Liver and gut tissues were homogenized in 2 volumes of ice cold water and aliquots of the homogenate were solubilized in a quaternary ammon ium tissue solubilizer (NCS, Amersham/ Searle Corp., Arlington Heights, IL) at 50°.The collected feces were added to the gut contents and the mixture was homogenized and aliquots of the homog enate were also solubilized. The metabolic chambers were washed with hot water to collect all urine adhering to the inner walls and floor. Aliquots of solubilized liver, gut homogenates, gut contents and urine were then added to a liquid scintillator solution (PCS, Amersham/ Searle Corp., Arlington Heights, IL) and the radioactivity measured in a liquid scintillation spectrometer. Radioactivity recovered in each tissue was expressed as a percentage of the administered dose. In the second experiment, 10 fed and 10 fasted rats (100 ±16 g) were used. Five of the fed rats and five of the fasted rats were each injected via tail vein with 8 piCi of
Downloaded from jn.nutrition.org by guest on July 14, 2011
(AMP) and guanosine monophosphate (GMP) to xanthine and uric acid during ab sorption. The conversion of the nucleotide to base and to uric acid was partially con firmed by Berlin and Hawkins (5) and by Khan (6) who showed catabolism of xanthine and hypoxanthine to uric acid in vitro. The final metabolic fate of exogenous purines in vivo is not well understood, except that allantoin is the major urinary end product of orally administered adenine in both fed and fasted rats (2). The chemical identity of the urinary end prod ucts of intravenously administered adenine is not well characterized. In addition, there is evidence that some exogenous adenine is converted to 2,8-dioxyadenine when large amounts are fed or in jected (7). The present studies were undertaken to evaluate the metabolism of orally administered adenine, guanine, hypoxan thine and xanthine in fed and fasted rats and to compare these results with the metabolism of the same purines when given by intravenous injection to iden tical rats. In addition, a comparison of the urinary end products of metabolism of orally and intravenously administered adenine was made to determine if the route of administration influences its overall catabolism. Finally, intestinal sacs were incubated with each of the four purines to determine the extent of trans port of unaltered free bases across the mucosal to serosal barrier. The results from these studies show that the gastro intestinal tract influences the absorption and metabolism of dietary purines.
ORALLY AND INTRAVENOUSLY ADMINISTERED PURINES
vein. The animals were placed in meta bolic cages and during the next 24 hours, complete urine collections were made. Urine collections were pooled within oral and intravenous treatments. The pooled urine from each treatment was immedi ately lyophilized, dissolved in 5 ml of water and centrifuged, and the clear super natant was layered on a 110 x 2.5 cm column of Sephadex G-10 (Pharmacia Fine Chemicals, Piscataway, NJ) and eluted with water. The column eluant was collected in 3.5 ml fractions and the optical density at 260 nm was monitored. A 100 ¡Aaliquot was withdrawn from each fraction and mixed with 10 ml of scintil lation fluid (PCS, Amersham-Searle Corp., Arlington Heights, IL) and counted in a liquid scintillation spectrometer. A 0.2 ml aliquot was also withdrawn from each op tically active fraction and its uric acid content was measured by the enzymatic spectrophotometric method (10). Fractions from the Sephadex G-10 column within each radioactive peak were pooled and lyophilized. The pooled, lyophilized radioactive materials were rechromatographed on the following sys tems: a) silica gel (Type G, Sigma Chemical Co., St. Louis, MO) developed in n-butanol:acetone:NH4OH:water in the volume ratios of 50:40:5:15; b) cellu lose developed in water and c) alumina developed in water (Catalog #13255 cellulose and 6062 alumina without fluorescent indicator, Eastman chromatogram sheets, Eastman Kodak, Rochester, NY; d) paper developed in water and e) paper developed in n-butanol:acetic acid:water in the volume ratios of 120: 30:50 (Whatman #1 chromatogram paper, Whatman Ltd., England). The pooled, lyophilized radioactive material from peak 3, because of its strong ultra violet (UV) absorption, was located under UV light in all Chromatographie systems. Areas of the silica, cellulose and alumina plates containing materials from peaks 5 and 7 were sprayed with p-dimethylaminobenzaldehyde, and the paper chromatograms containing materials from peaks 5 and 7 were sprayed with silver nitrate in bromophenol. Radio active areas on each chromatogram were
Downloaded from jn.nutrition.org by guest on July 14, 2011
[8-I4C]adenine while the remaining rats were each orally intubated with 8 /¿Ciof [8-14C]adenine. The rats were placed in individual metabolic cages and urine and feces were collected during the next 24 hours. At the end of this period, the rats were killed by decapitation and blood was collected in tubes containing sodium ethylenediaminetetracetate (EDTA). The liver, kidneys, spleen, heart and entire gastrointestinal tract (including contents) were quickly removed. Blood samples were centrifuged at 1,000 x g for 10 minutes. The erythrocytes were isolated and aliquots were solubilized as in experiment 1 and, in addition, were decolorized with a 5% solution of benzoylperoxide in toluene. The gastrointestinal contents were separated as in experiment 1. All tissues including carcasses were ho mogenized, solubilized and radioactivity measurements made and expressed as in experiment 1. In the third experiment, 12 fasted rats (140 ±40 g) were used. Five of the fasted rats were injected intravenously via tail vein with 12 juCi of [8-HC]adenine while the remaining seven rats were orally intubated with 12 /nCi of [8-14C]adenine. Both groups of rats were placed in individual metabolic cages and urine and feces were collected during the next 24 hours. The rats were then anesthetized with ether. Samples of blood, adipose tissue, lymph nodes, gastrocnemius mus cle, pituitary, salivary glands, thymus, thyroid and liver were promptly taken. All tissue samples were homogenized, solubilized and radioactivity measure ments made as in experiments 1 and 2. Recovered radioactivity was expressed as a percent of the administered dose re covered per gram fresh weight of tissue. Values from intravenously injected rats were divided by the corresponding values from orally intubated rats to obtain an intravenous/oral ratio. Determination of urinary end products. In the fourth experiment, eight fed rats (150 ±10 g) were used. Two hours after feeding, i.e. 1000 hours, 12 (¿Ciof [814C]adenine was given to each of four rats by gastric intubation and to the re maining four rats by injection into the tail
1795
1796
SAVAIANO ET AL.
the mixture from peak 3 was horizontally streaked on the chromatogram. The paper was dried. The UV absorbing strip with the same Rf value as standard uric acid was cut out and eluted by descending chromatography in the same solvent. The eluted material was dried and identified by IR spectroscopy. After IR measure ment, the solid potassium bromide disc containing the isolated material from peak 3 or standard uric acid was dissolved in water and the UV absorption spectrum was obtained for further identification. Intestinal sac incubations. In the fifth experiment, fasted rats (100 ±16 g) were anesthetized with ether and five 10-cm sections of upper small intestine, starting 6 cm distal to the pyloric sphincter, were removed from each rat. One end of each segment was closed with surgical thread. The intestinal sacs were loaded with 0.7 ml of modified Krebs-Heinseleit original ringer bicarbonate (KHRB) buffer (11) alone or supplemented with 28 /u.mol of adenine, guanine, hypoxanthine or xanthine individually. The KHRB buffer was modified by deleting calcium chloride, adding 200 mg glucose per 100 ml and aerating it with oxygen (95%) and carbon
TABLE 1 Twenty-four hour recovery radioactivity from tissues and excreta of fed and fasted rats given a single intravenous injection of [8-l4C]adenine, nuanine, hypoxanthine or xanthine in experiment 1' Tissue
Adenine
Guanine
UrineFedFastedGutFedFastedGut
Hypoxanthine
Xanthine
±70.98 ±0.21
3.46"±5.29"± ±82.55 ±0.07
±0.20 ±0.30
0.07"± ±0.03 0.04"±0.15"±±0.22
±0.15 ±0.10
±0.08 0.01»±0.85"± ±0.03
±0.18 ±0.50
0.74"±
Downloaded from jn.nutrition.org by guest on July 14, 2011
located with a thin-layer radioscanner (VarÃ-an Aerograph, Palo Alto, CA). The migration characteristics of the radio active compounds in peaks 3,5 and 7 were expressed in relation to the solvent front as Rf values. The radiolabeled com pounds were identified by comparing the Rf values in each system with those of known compounds. Additional aliquots of the pooled lyophilized material in peaks 5 and 7 from the Sephadex G-10 column were also studied by sublimation under reduced pressure at 70-75°.The sublimed material from peaks 5 and 7, as well as the residue after sublimation, were assayed for radio activity. The sublimed materials from peaks 5 and 7, as well as the sublimation residue from peak 5, were further identi fied by infrared (IR) spectroscopy (using potassium bromide) and melting point determination. Isolation of the radioactive compound in peak 3 from UV absorbing and/or fluorescent materials was achieved using the following paper Chromatographie method. An ascending paper chromatogram was developed in solvent (n-butanol; acetone:NH4OH:water; 50:40:5:15) after
fecesFedFastedLiverFedFastedGut content +
±0.03 ±0.30
content+ + gut fecesFedFasted23.0423.744.483.420.280.325.235.484.773.74±±-t--t-±-t--i--f-±±3.88"4.27"0.46»0.77*-"0.09"0.15a2.05»2.10a0.53a0.78 0.21"± ±0.34 ±0.11 0.05"78.55 ±8.45"17.69"0.05"'
