purine) nucleosides into hepatic RNA

The absorption and metabolism of dietary nucleic acids have received less attention than those of other organic nutrients, largely because of methodological difficulties. We supplemented the rations of poultry and mice with the edible alga Spirulina platensis, which had been uniformly labeled with '3C by hydroponic culture in 13CO2. The rations were ingested by a hen for 4 wk and by four mice for 6 days; two mice were fed a normal diet and two were fed a nucleic acid-deficient diet. The animals were killed and nucleosides were isolated from hepatic RNA. The isotopic enrichment of all mass isotopomers of the nucleosides was analyzed by selected ion monitoring of the negative chemical ionization mass spectrum and the labeling pattern was deconvoluted by reference to the enrichment pattern of the tracer material. We found a distinct difference in the 13C enrichment pattern between pyrimidine and purine nucleosides; the isotopic enrichment of uniformly labeled [M + 9] isotopomers of pyrimidines exceeded that of purines [M + 10] by >2 orders of magnitude in the avian nucleic acids and by 7and 14-fold in the murine nucleic acids. The purines were more enriched in lower mass isotopomers, those less than [M + 3], than the pyrimidines. Our results suggest that large quantities of dietary pyrimidine nucleosides and almost no dietary purine nucleosides are incorporated into hepatic nucleic acids without hydrolytic removal of the ribose moiety. In addition, our results support a potential nutritional role for nucleosides and suggest that pyrimidines are conditionally essential organic nutrients. The total daily requirements from all sources for purines and pyrimidines in human adults have been estimated to range between 450 and 700 mg/day (1, 2). The metabolic need for these nucleosides in healthy individuals is presumed to be met by de novo synthesis, although there is little in vivo information to support this belief. In addition to de novo synthesis of both the base and monosaccharide portions of the nucleosides, many cells possess efficient mechanisms for the recovery of nucleosides after hydrolytic breakdown of nucleic acids. The existence of these pathways, in principle, enables the use of nucleotides or nucleosides ultimately derived from the diet. For example, the cells of the intestinal mucosa have been shown to use preformed purine and pyrimidine bases (3), and supplementation of the diet with nucleosides has been shown to advance the growth and maturation of the developing small intestine (4). Whether there is a dietary "requirement" for nucleosides remains an open question. Little evidence is available to suggest that de novo synthesis and salvage pathways are inadequate in healthy well-nourished individuals to replace the daily losses of these molecules. Nevertheless, cellular immunity is decreased in animals that consume purified nucleotide-free diets (5). Resistance to infection in such animals is reduced and tolerance of allografts is increased. The absorption and utilization of nucleosides present in the diet, however, have proved difficult to estimate in a formally quantitative manner. We have recently used a method based on the ingestion of a uniformly 13C-labeled alga that enables us not only to identify the incorporation of intact dietary organic nutrients but also to detect the synthesis of nutritionally dispensable amino and fatty acids (6). In the present paper, we report the application of this method to investigate the absorption and metabolism of dietary nucleic acids. The results indicate that dietary pyrimidines and purines have different metabolic fates and are incorporated into hepatic nucleic acids to markedly different extents. MATERIALS AND METHODS Tracer Material. The prokaryotic blue-green alga, or cyanobacterium, Spirulina platensis, is a ubiquitous microorganism (7, 8), which contains 60-65% protein, 11% lipids, 15% carbohydrates, and <5% nucleic acids in its dry matter when grown in the laboratory (8-10). RNA has been reported to represent 2.2-3.5% of the dry weight, whereas DNA represents 0.6-1% (10-12). In the present study, the alga was grown in a closed system bioreactor under conditions in which the sole source of carbon was highly enriched (>99%) 13C02 (11, 12). All organic molecules in the resulting biomass, including the nucleosides, are virtually uniformly 13C labeled. Animal Feeding Trials. Chicken experiment. The uniformly labeled S. platensis was combined with corn and other dietary components to produce chicken feed. The composition of the feed was as follows (by weight): corn, 77.26%; salt, 0.12%; limestone, 4.016%; dical, 2.72%; vitamin premix, 0.033%; trace mineral premix, 0.033%; choline chloride, 0.087%; soybean oil, 1.87%; S. platensis (lyophilized alga), 13.83%; tryptophan, 0.025%. The feed was offered to a laying hen (DeKalb White Leghorn) for 27 days. The average feed intake was 100-120 g per day. At the end of the feeding period, the hen was killed, and its internal organs and carcass were individually deep frozen at -70°C. Data on the labeling of plasma and protein-bound amino acid labeling from this animal have been reported (6). Mouse experiment. Four mice (CD57, Charles River Breeding Laboratories) ("20 days of age) had free access for 3 days to feed that contained the following (by weight): basal purified diet (Purina), 89.0%; S. platensis (unlabeled), 10.0%; adenosine, guanosine, cytidine, uridine, and thymidine, each 0.2%. For the next 6 days, unlabeled Spirulina was replaced by Abbreviation: TMS, trimethylsilyl. tTo whom reprint requests should be sent at the present address: Abteilung fur Klinische Pharmakologie, Medizinische Klinik der Universitat Bonn, Sigmund-Freud-Strasse 25, Bonn 53105, Germany. 10123 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10124 Biochemistry: Berthold et al. uniformly 13C-labeled S. platensis. At the same time, the nucleoside supplementation was removed from the feed of two mice and replaced by an equal amount of the nucleoside-free basal feed. All mice received and completely consumed 3.3 g of feed per day. The composition of the basal diet was as follows (by weight): solca floc, 3.0%; casein, 21%; sucrose, 15%; corn oil, 5%; lard, 5%; choline chloride, 0.2%; dextrin, 43.65%; PMI vitamin mix, 2.0%; PMI mineral mix no. 10, 5.0%; DL-methionine, 0.15%. After 6 days, the animals were killed and their internal organs and carcasses were individually frozen at -70°C. Isolation of Liver RNA. High molecular weight RNA was isolated essentially as described (13), except that antifoam A (0.3%, vol/vol) was added to the lysis buffer. The initial RNA pellet was redissolved in lysis buffer and the extraction and precipitation steps were repeated. Finally, the RNA pellet was washed twice with ice-cold 75% isopropanol. Enzymatic Digestion of RNA. Dried RNA pellets were dissolved in autoclaved diethyl pyrocarbonate-treated water, and duplicate 100-,ug samples were adjusted to a concentration of 2 ,ug/ml and digested to nucleotides as described (14). The amounts of nuclease P1 and alkaline phosphatase (Sigma) were decreased 4-fold and digestion time was increased proportionately. Completeness of digestion was assessed by HPLC analysis (15) of a 2-,ug aliquot. Samples were dried in a Speed-Vac (Savant). Trimethylsilylation of Nucleosides and Bases (16). Nucleosides and purine and pyrimidine bases were purchased from Aldrich in the highest purity available (97-99+%). N,OBis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane (BSTFA/1% TMCS) was obtained from Pierce. Pyridine was obtained from Sigma. Standards of known concentration were dissolved in a mixture of BSTFA/1% TMCS and pyridine (5:1, vol/vol), Vortex mixed, and heated for 1 h at 100°C. After cooling, the samples were ready for mass spectrometric analysis. Nucleic acid digests were evaporated to dryness in a Speed-Vac and derivatized in the same manner. Amino Acid Isotopic Analysis (6). Hepatic protein was isolated by precipitation with 0.5 M perchloric acid. The dried protein pellet was hydrolyzed with 5.4 M HCl at 114°C for 24 h. The amino acids were derivatized with acid isopropanol and heptafluorobutyric anhydride and were analyzed by negative chemical ionization gas chromatography/mass spectrometry. Gas Chromatography/Mass Spectrometry. Gas chromatography/mass spectrometry was performed on a HewlettPackard HP 5988A system equipped with a HP 5890 II series gas chromatograph (Hewlett-Packard). The temperatures of the ion source, transfer line, and glass injection liner were all 250°C. One microliter of derivatization solution was injected (splitless) onto a 30m x 0.32mm DB-5 column (film thickness, 1 ,um) and eluted with helium as carrier gas. Mass spectrometry was performed by chemical negative ionization using methane as reagent gas at a pressure of 0.6-0.8 torr (1 torr = 133.3 Pa). Samples were measured in triplicate by selected ion monitoring of the trimethylsilyl ([TMS]4) molecular ions of adenosine and guanosine and the [M H]ions of uridine [TMS]3 and cytidine [TMS]4. Ion intensities were obtained through the full mass range of the compound from the unlabeled isotopomer [M] to the uniformly labeled isotopomer [M + x], where x is the number of carbon atoms of the underivatized molecule. Data Evaluation. The growth of the photosynthetic alga on 13C02 produced a biomass in which the majority (-85%) of the nucleosides were uniformly labeled. The present investigation then is, in effect, a multitracer study involving the introduction of (x + 1) labeled species, where x is the number of carbon atoms in the nucleoside in question. Because of the natural abundance of isotopes of the substituent elements, the enrichment of any single ion contains variable contributions of x + 1 ions so that expression of the results in terms of molar enrichments (i.e., ratios of tracer/tracee) requires the deconvolution of the overlying mass spectra. This, in its turn, involves the solution of x + 1 simultaneous linear equations of the general form