NUCLEOSIDES AND NUCLEOTIDES OF THE COLD ACID-SOLUBLE PORTION OF THE BLOOD OF STEERS 1

NUCLEOSIDES AND NUCLEOTIDES OF THE COLD ACID-SOLUBLE PORTION OF THE BLOOD OF STEERS 1 Robert C. Smith and Connie M. Stricker Auburn University 2, Auburn, Alabama 36830 SUMMARY The ultraviolet absorption spectrum of the cold acid-soluble portion of the blood of steers had a maximum absorption at 297 nanometers. This unusual spectrum was due to the large amount of uric acid riboside that was present. It accounted for over 96% of the material absorbing at 290 nm and for about 50% of the material absorbing at 260 nanometers. Other ultraviolet absorbing compounds present in the acid-soluble fraction were uric acid, AMp3, NADP, ADP, uric acid ribotide, ATP, GTP, UTP and unidentified compounds of UDP. (Key Words: Steers, Blood, Nucleosides, Nucleotides, Uric Acid Riboside.) INTRODUCTION The nucleosides and nucleotides of erythrocytes are important as substrates and cofactors in many reactions of the red blood cell. Adenosine triphosphate is especially important because it is necessary for the phospborylation of sugars in the initial reaction of glycolysis, it is important in cation transport, and it is required for the maintenance of the normal shape of the erythrocyte. Adenosine triphosphate must be maintained at a reasonable level for the erythrocyte to survive during its in vivo life span and during in vitro storage (Brewer, 1969). Since various diseases cause the concentration of the adenine nucleotides in the blood to deviate from normal, it was suggested that variations in these nucleotides might give some 1This investigation was supported by Hatch and State funds of Auburn University Agricultural Experiment Station. 2 Department of Animal and Dairy Sciences. 3Abbreviations used are: AMP--adenosine monophosphate,adp-adenosine diphosphate, ATP--adenosine triphosphate, NAD--nicotinamide-adenine dinucleotide, NADP--nicotinamide-adenine dinucleotide phosphate, GTP--guanosine triphosphate, UTP-uridine triphosphate, CTP--cytosine triphosphate, UDPuridine diphosphate, UMP--uridine monophosphate. indication of the metabolic condition of cattle (Baird, 1966). Although the concentrations of nucleotides are well documented for human blood, there are only limited data available for bovine blood. Bartlett (1970) has reported the elution pattern from a Dowex 1 column of the watersoluble phosphate compounds from the erythrocytes of a cow. Both 2,3-diphosphoglycerate and ATP were present in low concentrations when compared to man and other animals. Brown et al. (1972) reported the nucleotide profile separated by high pressure liquid chromatography of the blood of a cow. Baird (1966) assayed the concentrations of AMP, ADP, and ATP in the blood of dairy cows by enzymatic methods. In each of these studies only a few nucleotides were identified or quantitated. The present study was initiated to make a more thorough analysis of the nucleosides and nucleotides of the blood of steers. MATERIALS AND METHODS Four Angus steers (16-17 months of age) fed a 30% roughage-70% concentrate ration until the time of slaughter were used in this study. When the jugular vein was slit, duplicate 100 ml samples of blood were collected and immediately added to 300 ml of ice-cold 6.25% trichloroacetic acid (TCA). After 10 to 20 rain the extract was centrifuged at 12,000 g for 10 rain and the supernatant solution removed. The TCA was removed by extracting it five times with equal volumes of cold ether and freed from ether by aeration. The extract was lyophilized and the dried residue was dissolved with three 10-ml portions of water. The solutions were combined and adjusted to ph between 7 and 8 with a few drops of ammonium hydroxide. The extract was applied to a Dowex 1 8 column (200-400 mesh, formate form, 1 x 15 cm). After absorption of the extract the column was washed with water until the absorbance of the effluent was less than.1. The 1674 JOURNAL OF ANIMAL SCIENCE, Vol. 41, No. 6, 1975

ULTRAVIOLET-ABSORBING COMPOUNDS IN STEER BLOOD 1675 nucleotides were eluted with a gradient of formic acid and ammonium formate (Hudbert et al., 1954). The column was monitored continuously at 254 nm with an LKB absorbtiometer. Fifteen-milliliter fractions were collected and read at 260, 280 and 290 nm with a Beckman DU spectrophotometer. The concentration of nucleotides and uric acid riboside was estimated from their molar extinction coefficients (Laster and Blair, 1963; Pabst, 1956)9 The values are given as the mean +- SE. The ultraviolet absorbing compounds were identified by (a) comparison of the elution patterns obtained with those in the literature (Hudbert et al., 1954); (b) chromatography of the fractions on Whatman 3MM paper; (c) elution of the compounds from the paper chromatogram with water and comparison of their spectra with those in the literature (Pabst, 1956); and (d) hydrolysis of the nucleotides to the free base (Block et al., 1965). With the exception of uric acid riboside and uric acid ribotide, the compounds were chromatographed with authentic nucleotides purchased from Sigma Chemical Co., St. Louis, Missouri. The R F values of the uric acid compounds were compared with those in the literature. Compounds such as ATP that were easily identified were only chromatographed in one or two solvents9 Some of the compounds were chromatographed in five or six solvents. All chromatographic data are in table 1. RESULTS AND DISCUSSION The ultraviolet absorption spectrum of the acid-soluble portion of the blood of steers was unusual because the wavelength of maximum absorption was 297 nm (figure 1) rather than 260 nm, which is that normally found for blood of other species9 This unusual spectrum was due to the large amount of uric acid riboside present in the steer blood (Davis et al., 1922). An elution pattern of the cold acid-soluble material from steer blood absorbing at 260 and 290 nm is given in figures 2A and 2B. The first major fraction was the material not adsorbed to TABLE 1. SYMMARY OF CHROMATOGRAPHIC DATA FOR THE ULTRAVIOLET-ABSORBING COMPOUNDS IN THE COLD ACID-SOLUBLE PORTION OF STEER BLOOD Compound R F value Solvent: a A B C D E F Uric acid riboside.39 Uric acid riboside-literature Uric acid 121" Authentic uric acid.21 Uric acid ribotide.19 Uric acid ribotide-literature AMP 146 Authentic AMP.4-6 ADP.36 Authentic ADP.37 ATP.26 Authentic ATP.26 GTP... Authentic GTP... UTP... Authentic UTP NADP is Authentic NADP.29.50.48 b 9.37 9 9 53 c 22s 9 26 c 9.. 9.61.27.60 d.26 c.41.10.39.13.79.46.46 c.75....74....78....87....40.., o /i.33.63.65.12.12 asolvents of following composition (by volume): A, isobutyrie acid: NH,OH:H 20 (66:1:33); B, n-propanoh 1% NH4OH (3:2); C, n-propanoh2% NH4OH (3:2); D, 5% Na2 HPO 4 saturated with isoamyl alcohol, aqueous layer; E,.1 M phosphate (ph 6.8): ammonium sulfate:n-propanol (100:60:2); and F, isopropanohhchh20 (170:41:39). Compounds were detected with a Mineralight ultraviolet lamp (maximum emission, 254 nm). bdata from Forrest et al. (1961). CData from Hatfield et al. (1964). ddata from Laster and Blair (1963).

1676 SMITH AND STR1CKER w o n m 1.o CLS O.E o.4 0.2 I --. I ~. 240 260 211iO 300 1 320 chromatography where it was identified by reaction with.2% 2,6-dichloroquinone-4-chloroimide in 95% ethanol to form an orange color. When individual tubes of fraction 2 were lyophilized and chromatographed, uric acid riboside was present in all of the tubes. Uric acid was present mainly in the first two tubes and AMP in the last three tubes of this peak. Nicotinamide-adenine dinucleotide which is normally in this fraction was not detected, but the large amounts of uric acid riboside may have masked it. The third fraction contained NADP which was identified by chromatography and the formation of adenine on acid hydrolysis. It was also characterized by its ultraviolet absorption spectra (Pabst, 1956) and its reaction with 1 M NaCN to form a second maximum at 324 nm (Kaplan et al., 1955). There were 1.32 +.12 pmotes of NADP per 100 ml of blood. WAVELENGTH, nm Figure 1. Ultraviolet-absorption spectrum of the cold acid-soluble fraction of steer blood. the column. This fraction usually smeared when it was chromatographed on paper and the components were not identified. Uric acid riboside was the major ultravioletabsorbing compound in fraction 2. It was identified by its ultraviolet absorption spectra at acid, alkaline, and neutral ph (Forrest et al., 1961), its reaction with.2% 2,6-dichloroquinone-4-chloroimide in 95% ethanol to form a yellow product on paper chromatograms (Forrest et al., 1961), its R F values and its hydrolysis to uric acid by heating at 100 C for 2 hr in 3 N HC1. After the uric acid riboside was purified by paper chromatography, the ratio of uric acid to ribose was 1. Uric acid riboside accounted for over 50% (43 to 56%) of the material absorbing at 260 nm (figure 2A) and for over 96% of the material absorbing at 290 nm (figure 2B). There were 58.5 + 1.2 /2moles of uric acid riboside per 100 ml of blood. Up to 30 /~moles of uric acid riboside have been crystallized from 100 ml of blood (Forrest et al., 1961; Newton and Davis, 1922). In addition to uric acid riboside, this peak also contained AMP and uric acid. The AMP was identified by chromatography, ultraviolet absorption spectra (Pabst, 1956), and hydrolysis to adenine. Uric acid was characterized by its spectra and E r 0 S 6t t~ 4 UJ 0 3 se m 2 a: 0 W m 1 E 0 5 Q 4 ud c~ 3 z.- 2 0 20 40 60 F R A C T I O N S (15 ml) Figure 2. Chromatogram of the cold acid-soluble fraction of steer blood. (A) Absorbance at 260 nm; (B) Absorbance at 290 nanometers. S

ULTRAVIOLET-ABSORBING COMPOUNDS IN STEER BLOOD 1677 The fourth fraction contained ADP and uric acid ribotide. Adenosine diphosphate was identified by its spectra (Pabst, 1956) and R F values. Uric acid ribotide was identified by its spectra (Hatfield et al., 1963), its R E values, its reaction on the chromatograms with 2,6-dichloroquinone-4-chloroimide and subsequent spraying with 5% Na2HPO4 to give a yellow color (Hatfield et al., 1963), and the production of uric acid by heating in 3 N HCI at 100 C for 1 hour. There were 5.16 -+.63 /~moles of ADP and 1.24 +.10/~moles of uric acid ribotide per 100 ml of blood. The ratio of uric acid riboside to uric acid ribotide was about 47:1. Values of 77:1 (Hatfield et al, 1963) and 300:1 (Hatfield and Forrest, 1962) have been reported previously. In some experiments the ADP peak was much lower than in others. When this occurred there was no corresponding increase in ATP that would account for the change. Adenosine diphosphate was the only fraction that varied in this way. The next few minor fractions were not identified, but they had ultraviolet spectra that suggested that they were uracil compounds. Uracil was the only base located on chromatograms after these fractions were hydrolyzed in 12 N HC104 at 100 C for 1 hour. Fraction 5 contained ATP which was identified by its spectra (Pabst, 1956), acid hydrolysis to adenine, and chromatography. There were 9.15 -+.40 gmoles of ATP per 100 ml of blood. Values of 5.0 (Brown et al., 1972), 8.0 (Bartlett, 1970) and 13.1 (Baird, 1966) gmoles per 100 ml of blood have been reported. Although it appeared from the slight skewing of the ATP peak and the 280/260 ratio (.36) of this area that other compounds might be present, only ATP was recovered when this fraction was adsorbed to carbon, eluted, and then chromatographed. Although erythrocytes from some animals contain CTP, the levels of the cytosinecontaining nucleotides are extremely low (Bartlett, 1970; Mandel, 1964). They were not detected in the blood of the steers used in this study. The last fraction contained both GTP and UTP which were identified by chromatography and by acid hydrolysis to guanine and UMP. The GTP was also characterized by its typical blue fluorescence in the isopropanol solvent. The concentration of GTP plus UTP was.84 -+.06 gmoles per 100 ml of blood. Since this study was completed, Leyko et al. (1974) reported that the adenine nucleotides of the erythrocytes of Polish Red and Charolais bulls were lower than those of Black and White Lowland bulls. It is hoped that further studies of the blood concentration of uric acid riboside and nucleotide profiles of animals of different ages, breeds and nutritional status will provide some insight into the metabolic status of cattle. LITERATURE CITED Baird, G. D. 1966. Blood adenine nucleotide levels in the normal and the acetonaemic dairy cow. Nature 210:863. Bartlett, G. R. 1970. Patterns of phosphate compounds in red blood cells of man and animals. In G. J. Brewer (Ed.) Advances in Experimental Medicine and Biology. Vol. 6. Plenum Press, New York. Block, R. J., E. L. Durrum and G. Zweig. 1965. Paper Chromatography and Electrophoresis. Academic Press, Inc., New York. Brewer, G. J. 1969. Adenosine triphosphate. In J. J. Yunis (Ed.) Biochemical Methods in Red Cell Genetics. Academic Press, New York. London. Brown, P. R., R. P. Agarwal, J. Gell and R. E. Parks. 1972. Nucleotide metabolism in the whole blood of various vertebrates: Enzyme levels and the use of high pressure liquid chromatography for the determination of nucleotide patterns. Comp. Biochem. Physiol. 43B:891. Davis, A. R., E. B. Newton and S. R. Benedict. 1922. The combined uric acid in beef blood. J. Biol. Chem. 54:595. Forrest, H. S., D. Hatfield and J. M. Lagowski. 1961. Uric acid riboside. Part I. Isolation and reinvestigation of the structure. J. Chem. Soc. 963. Hatfield, D. and H. S. Forrest. 1962. Biosynthesis of 3-ribosyluric acid (uric acid riboside). Biochim. Biophys. Acta 62 : 185. Hat-field, D., R. A. Greenland, H. L. Stewart and J. B. Wynngaarden. 1964. Biosynthesis of a new uric acid ribonucleotide. Biochim. Biophys. Acta. 91:163. Hatfield, D., R. R. Rinehart and H. S. Forrest. 1963. 3-Ribosyluric acid. Part II. Isolation of the corresponding nucleotide from beef blood. J. Chem. Soc. 899. Hurlbert, R. B., H. Schmitz, A. F. Brumm and V. R. Potter. 1954. Nucleotide metabolism. II. Chromatographic separation of acid-soluble nucleotides. J. Biol. Chem. 209:23. Kaplan, N. O., M. M. Ciotti, F. F. Stolzenbach and N. R. Bachur. 1955. Isolation of a DPN isomer containing nicotinamide riboside in the ~ linkage. J. Amer. Chem. Soc. 77:815. Laster, L. and A. Blair. 1963. An intestinal phosphorylase for uric acid ribonucleoside. J. Biol. Chem. 238:3348. Leyko, W., G. Bartosz, W. Duda, Z. Wojtkowiak, and A. Kolataj. 1974. The content of nucleotides in erythrocytes of different breeds of cattle. Genet. Polon. 15:91. Mandel, P, 1964. Free nucleotides in animal tissues. In J. N. Davidson and W. E. Cohn (Ed.) Progress in Nucleic Acid Research and Molecular Biology. Vol.

1678 SMITH AND STRICKER 3. Academic Press, New York. London. Chem. 54-:601. Newton, E. C. and A. R. Davis, 1922. The distribution Pabst Laboratories. 1956. Ultraviolet absorption specof the combined uric acid in beef blood. J. Biol. tra of 5'-ribonucleotides. Circ. OR-IO.