Use of High Pressure Liquid Chromatography to Investigate the “ln Vitro” Reactions of Human Erythrocytes with Guanosine Phyllis R. Brown and R. E. Parks, Jr. Section of Biochemical Pharmacology, Division of Biological & Medical Sciences, Brown University, Providence, R. 1. 02972
High pressure liquid chromatography has made possible the separations of biologically important molecules in small samples of tissue with high sensitivity, speed, accuracy, and resolution. Because of the excellent separation of nucleotides, this technique is valuable in studying metabolic pathways. In pharmacology, the formation of drug metabolites can be monitored simultaneously with the effect of the drug on the naturally occurring nucleotide pools. Reported in this paper is the use of high pressure liquid chromatography to examine the “in vitro” reactions of human erythrocytes with guanosine. The reproducibility of retention times, ease, and versatility of operation and precision of results, which make this technique valuable, are demonstrated.
Before the development of high pressure liquid chromatography for determination of cellular nucleotide pools (1, 2), investigations of cell metabolism were hampered by the lack of good technology. Techniques such as column, paper, or thin layer chromatography, although useful, were time consuming or not sufficiently accurate or sensitive. Furthermore, excellent techniques such as enzyme cycling methods (3-5) permitted only the determination of a few nucleotides or a small group of nucleotides at one time. Often large tissue samples were required. Most investigators resorted to isotopic techniques, which while sensitive and accurate, could not always be used, especially in clinical studies. Therefore, high pressure liquid chromatography has fitted a particular need for those studying nucleotide metabolism. In the field of pharmacology, it is useful because it is possible to monitor simultaneously the formation af drug metabolites and the majority of the naturally occurring nucleotides. These analyses are rapid, sensitive, and quantitatively reproducible. Base-line studies have been made on the formed elements of human blood (6) so that they may be used as references in predicting or diagnosing certain disease states or in monitoring drug therapy. Each formed element had a characteristic nucleotide pattern and the concentrations of each nucleotide within the elements of normal subjects were highly reproducible. Other base-line studies have been made of species differences in the nucleotide pools of whole blood (7). Each species had a characteristic nucleotide profile and there was good reproducibility of the patterns within the species. Chromatograms ( 1 ) C. Horvath, B. Preiss, and S. Lipsky, Anal. Chern., 39, 1422 (1967). (2) P. R . Brown, J. Chrornatogr.,52, 257 (1970). (3) 0. H . Lowry, J. V. Passonneau, D. W. Schulz, and M . K. Rock, J . Biol. Chem., 236, 2746 (1961). ( 4 ) S. Chaand ‘2.4 M. Cha, Mol. Pharrnacoi., 1, 178 (1965). (5) S. ChaandC-J. M . Cha, Anal. Biochern., 33, 174 (1970). (6) E. M. Scholar, P. R. Brown, R. E. Parks, Jr., and P. Calabresi, Blood, in press. ( 7 ) P. R. Brown, R. P. Agarwal, J. Gell, and R . E. Parks, J r . , Cornp.
Biochern. Physiol. in press.
948
ANALYTICAL CHEMISTRY, VOL. 45, NO. 6 , M A Y 1973
of the UV absorbing constituents in urine are now available (8-10) as are nucleotide patterns of normal rat liver and brain (11, 12). Therefore, high pressure liquid chromatography was the method of choice used in a study of human erythrocytic metabolism. As a part of this overall investigation, the reactions of human erythrocytes with guanosine were examined. Although guanine nucleotides are normally present in low concentrations in human erythrocytes as compared to leukocytes (6) and as compared to the concentrations in the blood of other species (7), the fact that they are present suggests that they play a role in human erythrocytic metabolism. The enzymes of the metabolic pathway from guanine to GTP, i.e., hypoxanthine-guanine phosphoribosyl-transferase, guanylate kinase, and nucleoside diphosphokinase, have been demonstrated in human erythrocytes (13). The significance of guanosine and its nucleotides, however, in human erythrocytic metabolism has not been well established. Therefore, the reactions of guanosine in human erythrocytes were investigated.
EXPERIMENTAL Materials. Fresh blood in acid-citrate-dextrose (ACD) from which the platelets had been removed was obtained from the Division of Hematologic Research of the Pawtucket Memorial Hospital. The adenosine and inosine were purchased from P-L Biochemicals and the guanosine, hypoxanthine, and inosine from Sigma Chemical Co. The 2-fluoroadenosine was sent to us by H . Wood of the Cancer Chemotherapy National Service Center. Methods. Fresh blood was washed twice in isotonic saline and the buffy coat and supernatant fluid were discarded. Blood that had been stored in ACD medium for one month was used as indicated and the erythrocytes washed and separated in the same manner. A 33% suspension of erythrocytes was incubated in a medium that contained 128mM NaC1, 1.2mM MgC12, 18mM KH2P04 ( p H 7 . 4 ) , and 16mM glucose unless stated for specific experiments. Guanosine and other purines were added so that their final concentration in the solution was 0.5mM unless specifically stated. All incubations were carried out in a Dubnoff shaking water bath at 3’7 “C (80 oscillations/minj with air as the gas phase. In the preincubation experiments with F-AdR, the erythrocytes were incubated for 1 hr with the appropriate substrate.beforethe start of the incubation experiment. Sample Preparation. One milliliter of the suspension of erythrocytes was pipetted dropwise into 2 ml of cold 12% TCA and stirred rapidly on a Vortex mixer (14). After centrifugation, 0.5ml samples of the supernatant fluid were extracted with HzO-saturated diethyl ether. For the kinetic studies, 0.5-ml samples were removed from the erythrocytic medium 0, 5, 10, 20, 30, 60, 120,
C. D. Scott, J. Attrel. and N. G . Anderson, Proc. SOC.f x p . Biol. Med., 125, 181 ( 1 9 6 7 ) . C. D. Scott and N. E . Lee, J . Chromatogr., 42, 263 (1968) Scott, C. D. Ciin. Chem. 14, 521 (1968). P. R. Brown, “High Pressure Liquid Chromatography, Biochemical and Biomedical Applications,”Academic Press, New York. N.Y., in press. H. W. Shmukler, J. Chromatogr. Sci.. 10,38 (1972). R. P. Agarwal, E. M . Scholar, K. L . Agarwal. and R . E . Parks, Jr., Biochem. Pharm.. 20, 1341 (1971) R . P. Miech and M . C. Tung, Biochern. Med., 4,435 (1970)
0 min. ---- 10 min.
I
--20min. .......30min.
ATP
ADP
GDP
TIME IN MINUTES
Figure 1. Progressive synthesis of guanosine nucleotides in human erythrocytes Fresh washed human erythrocytes were incubated with guanosine (0.5mM) at 37 “C. Chromatograms are shaown of nucleotide extracts of aliquots that were removed from the reaction mixture at 0, 10, 20, and 30 min after the start of the incubation
180, and 240 min after the addition of the guanosine. Each sample was added to 1 ml of cold 12% TCA. Nucleotide Analysis. The high pressure liquid chromatography procedure used was based on that described previously (2). Samples (20 pl) were injected into a Varian Aerograph LCS 1000 equipped with a Reeve Angel column (1 mm X 3 m ) packed with a strong anion exchange pellicular resin Pellionex AS SAX. The eluents were 0.001M KHzPO4 (pH 4.5) and 0.25M KHzPO4 in 2.2M KC1 (pH 4.5). The column flow rate was 1 2 ml/hr and the gradient flow rate 6 ml/hr. The full scale absorbance was 0.04 O.D. unit. The peaks were identified by use of internal standards, by comparison to chromatograms of known standard nucleotides, and by enzymic peak-shift techniques. The concentrations of the nucleotides were determined by comparing peak areas (peak height times width a t half height) with those of standard solutions of known concentrations.
RESULTS AND DISCUSSION With high pressure liquid chromatography it was possible to monitor the “in vitro” reactions of human erythrocytes with guanosine rapidly, sensitively, and accurately. Prior to the development of this instrumentation, it had been difficult to measure the guanine nucleotides in human blood because of the low concentrations present. An excellent cycling method has been described by Cha and Cha (5) but this technique is applicable to the guanine nucleotides alone. To observe the dynamic changes in the majority of all the nucleotides in the cells was a time consuming and difficult task. When washed human erythrocytes from fresh blood were incubated with guanosine, GTP was formed rapidly and the G T P concentration was stable over the 4-hour incubation period. Because of the ease and simplicity of operation of the chromatograph, it was possible to follow the progress of the nucleotide synthesis as shown in Figure 1. With erythrocytes from stored blood, which had only Y3 the ATP concentration as that found in fresh blood, the maximum G T P concentration was lower than that produced with fresh blood and the GTP concentration was about the same as the ATP concentration present. The GTP was synthesized at a slower rate; maximum GTP concentration was formed at 1-hr incubation. An interesting observation was that on incubation of the stored blood with guanosine, there was a shift in the ATP:ADP ratio. Within 10 min, the large AMP and ADP peaks decreased significantly and the ATP increased, although the
I
I
,
.
20
40
60
TIME IN MINUTES
Figure 2. Synthesis of guanosine nucleotides in human erythrocytes from blood that had been stored for one month and incubated with guanosine at 37 “C Chromatograms are shown of nucleotide extracts of aliquots that were removed from the reaction mixture at 0 ( A ) , 10 (B). 30 ( C ) , and 120 ( D ) min after the start of the incubation
ATP concentration did not reach the ATP level found in fresh blood (Figure 2 ) . To examine possible metabolic pathways, guanosine and hypoxanthine were incubated with fresh erythrocytes. The same maximum concentration of GTP was formed when the hypoxanthine and guanosine were present together as with the guanosine alone but the rate of formation was reduced (Figure 3). With inosine and guanine, the 5’-monophosphate nucleotide of inosine accumulated as was shown previously ( 1 5 ) and the GTP formation reached a maximum a t 2 hr and then decreased (Figure 4). However, less IMP was formed than when the erythrocytes were incubated with inosine alone. Incubation with guanine alone or in combination with inosine produced about Yz the concentration of GTP as with guanosine. It will be interesting to learn whether a transport problem is involved. These experiments will be rerun, preincubating the cells with the bases, guanine and hypoxanthine. By building up a larger supply of the bases in the cells, it may be possible to determine more clearly the relative rate of nucleotide formation when the ribosides are added to the incubation mixtures. When fresh human erythrocytes were incubated with a combination of guanosine and adenosine or guanosine and inosine, the final GTP levels were the same but smaller amounts of AMP or IMP were produced than were noted (15) R. E.
Parks, Jr., and P. R. Brown Biochem., in press.
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 6 , M A Y 1973
949
---
ATP
- GTP
I
Time in minutes Figure 3. Plots of GTP and ATP concentrations vs. time of incu-
bation Fresh washed erythrocytes were incubated with 0 . 5 m M guanosine and 0.5mM hypoxanthine at 37 "C
2.0
t
-
C
AT P GTP IMP
20
O
L
-
J 60
120
I80
Time in minutes Figure 4. Plots of GPT, ATP, and I M P concentrations vs. time of incubation Fresh washed erythrocytes were incubated with 0.5mM guanine and 0.5mM inosine at 37 "C
previously on incubation of erythrocytes with adenosine or inosine alone (4). Incubation of human erythrocytes with adenosine or inosine results in accumulation of large amounts of the monophosphate nucleotides with no increase in the concentration of the triphosphate nucleotides. With 2-fluoroadenosine, however, the triphosphate nucleotide of the fluoroadenosine is produced readily in large concentrations under the same incubation conditions. FAdR, a highly cytotoxic agent with broad spectrum antibacterial activity, is readily anabolized in cells but is not catabolized (16). Therefore it was of interest to see whether this ribonucleoside affected the formation of GTP. The presence of F-AdR (0.25mM) inhibited the formation of guanosine nucleotides either on preincubation with FAdR or on incubation of guanosine in the presence of FAdR (Figure 5 ) . In all the experiments, the ATP concentrations were constant and no hemolysis of the erythrocytes was observed during the course of the incubation. The experiments described in Figures 1 and 3 were performed with a 33% suspension of erythrocytes and 0.5mM guanosine. It may be estimated that there occurred essentially complete conversion of the guanosine to the ribonu(16) J. A. Montgomery and K. Hewson, J. Med. Chem., 12, 498 (1969).
950
ANALYTICAL CHEMISTRY, VOL. 45, NO. 6, M A Y 1973
40 TIME IN MINUTES
60
Figure 5. Chromatograms of fresh human erythrocytes incubated for 2 h r with guanosine, fluoroadenosine, or a combination of guanosine and fluoroadenosine Fresh washed human erythrocytes were incubated with 0.25mM guanosine, 0.25mM fluoroadenosine, or a combination of 0.25mM fluoroadenosine and 0.25mM guanosine. The chromatogram of the nucleotide extracts of the reaction mixture with guanosine is shown in A, with fluoroadenosine in B, and the combination in C
t
GDP
10
20
30
40
GTP
50
60
70
TIME IN MIN.
Figure 6. Chromatogram of human erythrocytes incubated for 2 hr in the presence of a high concentration of phosphate buffer Fresh washed erythrocytes were incubated with 5.0mM guanosine in a medium containing 20mM phosphate buffer
cleotide level. This raised the question of whether it is possible for erythrocytes to form larger quantities of gua-
sensitivity, accuracy and rapidity as has been obtained with the nucleotides. Thus a more complete picture of human erythrocytic nucleotide metabolism can be formulated. Studies are also under way with a preparative column so that larger samples can be collected to characterize compounds whose peaks have not yet been positively identified.
nine nucleotides. In the studies of Mager et al. (1 7), it was shown that glucose and phosphate concentrations influenced the uptake of purine bases by human erythrocytes. In our studies with ribonucleosides, it was found that the formation of GDP and GTP took place without added magnesium or glucose in the incubation medium. The total concentration of GTP formed, however, was influenced by phosphate concentration. In the presence of excess guanosine (5mM) and low phosphate, the concentration of GTP formed was about 213 that of ATP. When higher concentrations of phosphate were present (20mM or greater) the GTP concentration exceeded by two-fold the concentration of ATP (Figure 6). We have not yet determined the maximal quantity of guanine nucleotides that can be formed in human erythrocytes but the amount synthesized in these experiments may be well described as remarkable. The availability of high pressure liquid chromatography will make possible many additional studies such as the influence of temperature, ionic composition of the medium, the role of nucleoside transport etc. on the rate of GTP synthesis in erythrocytes. Work is under way using high pressure liquid chromatography together with radioisotopic techniques to determine the exact pathways of these reactions. Techniques are being worked out to determine the free bases and the nucleosides in the cell using this equipment, with the same
The following abbreviations are used: AMP, ADP, ATP = adenosine S‘-mono-, 5’-di-, and 5’-triphosphate; GMP, GDP, GTP = guanosine 5’-mOnO-, 5’-di-, and 5’-triphosphate; IMP = inosine 5’-monophosphate; FAdR = fluoroadenosine; ACD = acid-citrate-dextrose; TCA = trichloracetic acid.
G . Izak, lsrael J.
Received for review November 22, 1972. Accepted January 29, 1973. Supported by Grant GM No. 16538 from the Public Health Service
( 1 7 ) J. Mager, A . Divilansky, A. Razin, E. Wind, and Med. Sci., 2, 297 (1966).
NOMENCLATURE
ACKNOWLEDGMENT The authors wish to thank Mrs. Sandra Bobick and Jonathan Gel1 for their fine technical assistance, Mrs. Andrea Bullard for the excellent illustrations, the Hematologic Research Division of the Memorial Hospital for kindly supplying us with fresh blood, and Harry Wood of the Cancer Chemotherapy National Service Center for the 2-fluoroadenosine.
ANALYTICAL
CHEMISTRY, VOL. 45, NO.
6, M A Y 1973
951
