BLOOD LEVELS OF ADENINE NUCLEOTIDES Harry G. Albaum, … , Theodore Cayle, Arthur Shapiro J Clin Invest. 1951;30(5):525-530. https://doi.org/10.1172/JCI102468. Research Article Find the latest version: https://jci.me/102468/pdf BLOOD LEVELS OF ADENINE NUCLEOTIDES 1 BY HARRY G. ALBAUM, THEODORE CAYLE, AND ARTHUR SHAPIRO (From the Biology Research Laboratory, Brooklyn College, Brooklyn, N. Y.) (Submitted for publication December 23, 1950; accepted, March 17, 1951) INTRODUCTION The procedure used in our work is a spectro- photometric assay on perchloric acid filtrates, using The metabolic significance of the adenine nucleo- in modified form the enzyme techniques of Kalckar tides (1, 2) and the reports of the therapeutic ac- (15-17). tion of muscle adenylic acid and adenosine triphos- In addition to presenting observations on the phate in various pathological states (3-12) has on a stimulated interest in the possible mechanism of ac- concentration of the various nucleotides group of four normal subjects (three men and one tion in these compounds. woman) over a period of about eight months, data Recently a rapid reliable method was developed will be presented on the effects of intramuscular by one of us (13) for measuring total adenine, injections of muscle adenylic acid, adenosine tri- adenylic acid (AMP), adenosine diphosphate and yeast acid on the levels of (ADP) and adenosine triphosphate (ATP). We phosphate adenylic have applied this method to the study of blood since the above compounds. it is the only human tissue which can be con- ANALYTICAL PROCEDURE veniently studied. Before studies could be undertaken on pathologic a) Estimation of total adenine bloods, we felt that normal bloods should be ex- Total adenine nucleotide was assayed by drawing blood, initially under oxalate, and immediately deproteinizing 1 amined for total nucleotide content as well as for ml. with 4 volumes of cold 2% perchloric acid. The pro- possible content of adenylic acid, adenosine diphos- tein precipitate was washed with an additional volume of phate and adenosine triphosphate. That adenine cold 2% perchloric acid. The combined supernatant solu- nucleotides are present in blood has been known tions collected after centrifugation were adjusted to pH since 1914. The first completely adequate quanti- 7.0 and made to a final volume of 10 ml. One ml. of fil- trate was added to a tube containing 3.5 ml. H20 and 0.5 tative data are those of Buell in 1935 (14) who es- ml. M/10 phosphate buffer at pH 7.4. The resulting solu- timated total nucleotide nephelometrically after tion when read in the spectrophotometer shows an ab- precipitating with uranium and then silver, from sorption maximum at 258 ms, corresponding to the ab- a neutralized trichloracetic acid filtrate. Apart sorption maximum of adenine nucleotides in perchloric from describing the procedure itself, Buell reported acid. Total nucleotide was calculated as adenine using that the nucleotides are present principally in the muscle adenylic acid as a standard. red cells (indeed the value varies with the hemato- b) Estimation of AMP, ADP and ATP crit); that the level for any one individual over a Adenylic acid, adenosine diphosphate and adenosine period of several months is fairly constant, and triphosphate were assayed in the same filtrates according that the average value for males is about 11.5 to the procedure described by Albaum and Lipshitz (13), mgm.%b and for females 10.0 mgm.%o (expressed in the following way: One ml. aliquots of the filtrate were placed in three separate tubes. Into the first tube 0.5 ml. as adenine). 0.1 M malonate buffer pH 5.9, 0.03 ml. of adenylic acid de- Our interest, as indicated above, was not only in .minase (containing 15 /Agm. protein N) and water to make the total nucleotide, but also in the constitution of a final volume of 5.0 ml. were pipetted and the change in the nucleotide. We also felt that the Buell pro- density in the spectrophotometer at 265 ms recorded at cedure for nucleotides was much too the end of 10 minutes. The second tube received buffer, estimating water, 0.1 ml. MgCl, (10 mgm./ml.), 0.01 ml. myokinase cumbersome. We, therefore, concentrated our at- (containing 4 ugm. protein N) and deaminase and again tention on the preparation of filtrates which could the change in density at 265 mnu was recorded at the end of be assayed directly. 10 minutes. To the third tube was added buffer, water, MgCl2, myokinase, 0.1 ml. 0.5 M glucose, 0.01 ml. hex- 1 This work was supported by a grant to Brooklyn Col- okinase (containing 7 sugm. protein N) and the deaminase lege from the Ernst Bischoff Company, Ivoryton, Conn. and once more the change in density determined. Any 525 5265HARRY G. ALBAUM, THEODORE CAYLE, AND ARTHUR SHAPIRO change in tube 2 over that occurring in tube 1 is due to TABLE II the presence of ADP. Since only 1 mole of AMP is Effect of standing on adenine nuceotides in blood formed from 2 moles of ADP, the difference between tubes 1 and 2 is multiplied by a factor of 2. The third % of total purine as tube records the change in density due to AMP, ADP and Temper- Time ature ATP. Since the concentration of AMP and ADP are ATP AMP ADP now known (by reading the adenine due to these com- pounds from a standard curve derived from the change in density of known concentrations of these Oxalated blood 0 68 0 0 nucleotides) 90 15 28 25 and the total adenine is known from tube 3, the difference Room between the AMP and ADP adenine and the total adenine Heparinized blood 0 58 0 0 gives the value for the ATP adenine. 90 59 0 4 The reactions discussed above are represented by the Oxalated blood 0 40 0 5 following equations: 90 12 20 7 370 C. adenylic acid deaminase Heparinized blood 0 39 0 5 (1) AMP - Inosinic Acid 90 44 0 0 myokinase (2) 2ADP -4 ATP + AMP hexokinase prepared according to the method of Kalckar (15); myo- (3) ATP + glucose - > ADP + glucose-6- phosphate kinase, according to Colowick and Kalckar (18); hexo- kinase was prepared in partially pure form according to Before experiments were undertaken on the distribution the method of Berger, Slein, Colowick and Cori (19) of the compounds, several preliminary runs were carried through step No. 3 (fractionation with ethanol at 00 C.) out to see whether the method as described was reliable, of the isolation procedure. The enzymes were kept in a i.e.,-could added nucleotide be recovered and measured? deep freeze and were found to be stable over long periods The results of one such experiment are shown in Table of time. I. The total nucleotide accountable for as AMP, ADP Muscle adenylic acid used for injection was supplied by and ATP in this sample of blood was 6.4 Igm. It is ap- the Ernst Bischoff Co., Ivoryton, Conn. as My-B-Den (1 parent that added ATP can be recovered indicating that ml. ampules containing 20 mgm. as the sodium salt). In the method is reliable. some experiments ampules containing 100 mgm./ml. were Early in our studies on the concentration of the various used. ATP was also obtained from the Ernst Bischoff nucleotide fractions in oxalated human blood, it was Co. and was administered as a solution of the sodium salt. found that ATP content decreased significantly within the first hour, though not in the first five minutes. For EXPERIMENTAL this reason, in the early work which was done on oxalated blood, the samples were pipetted into perchloric acid within Total nucleotide content of blood one minute of withdrawal. To avoid the inconvenience of this procedure, the stability of the nucleotide in heparin- The total nucleotide content of the blood of the ized blood was investigated. Table II shows the percent- four subjects used in the experiments expressed age of total purine found in the ATP, AMP and ADP as mgm. %o adenine are shown in Figure 1 (closed fractions, respectively, in freshly drawn blood, and in the circles). Several conclusions may be drawn from same blood samples after 90 minutes. The experiment was these data: 1.) While there is some variability in run at room temperature and at 37° C. in the presence of a mixture of potassium oxalate and ammonium oxalate, total nucleotide, the levels remain fairly uniform of which the oxalate concentration was 112 mgm. %, and over a period of about eight months. The variabil- in the presence of 5 mgm. % of heparin. It is clear that ity is no greater than one finds in the levels of other the ATP is very significantly split to ADP and AMP in blood constituents under normal conditions. 2.) oxalated blood, while it is quite stable in the heparinized blood. The levels obtained are similar to those obtained Adenylic acid deaminase (Schmidt's deaminase) was by Buell using the nephelometric procedure, so that we appear to be measuring the same thing that TABLE I Buell measured. Recovery of ATP when added to perchloric acid filtrate of blood Nucleotide present as AMP, ADP and ATP 1. Total AMP, ADP, and ATP found in blood sample = That portion of the total nucleotide accountable 6.4 ugm. for as AMP, ADP and ATP is also shown in Fig- 2. ATP added to blood sample = 8.2 %gm. 3. Total AMP, ADP, and ATP in blood and added ATP ure 1 (open circles). It is immediately clear that calculated (1 +2) = 14.6 t'gm. the level of these compounds shows considerably found = 14.3 ogm.