A sensitive radioimmunoassay for adenosine in biological samples

ANALYTICAL

BIOCHEMISTRY

A Sensitive

121,409-420

(1982)

Radioimmunoassay

for Adenosine

TOMOKAZU SATO,* AKIRA KUNINAKA,* *Research

in Biological

Samples

HIROSHI YOSHINO,* AND MICHIO UI?

Laboratories, Yamasa Shoyu Company, Ltd., Choshi. Japan, and TDepartment Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo.

of Physiological Japan

Received November 16. 1981

A simple, sensitive, specific, and reproducible radioimmunoassay for the measurement of adenosine in biological materials has been developed. Adenosine antibody was obtained by immunizing rabbits with an immunogen prepared by conjugating 2’,3’-disuccinyladenosine to human serum albumin. By succinylating adenosine in samples at the 2’- and 3’-0 positions with a premixed reagent consisting of succinic anhydride, triethylamine, and dioxane, the assay became sensitive enough to detect less than picomole amounts of adenosine in minute quantities of tissues. The cross-reactivity of structurally related compounds with the antibody was mostly negligible except for 2’-deoxyadenosine, whose usual concentration was very low. The use of this method made it possible to measure adenosine without any prior purification procedure. The immunoreactive materials in various biological samples disappeared during incubation of the samples with adenosine deaminase.

Adenosine has attracted much attention in the biological sciences, since a large number of reports have shown that adenosine is associated with immune response ( 1,2), coronary vasodilatation (3,4), neurotransmission (5), hormone secretion (6) cyclic nucleotide formation (7- 13) and other phenomena. Accordingly, it seems important to know not only the action of exogenously added adenosine but also the amount of adenosine in biological fluids and tissues in a physiological or pathological state. However, limited information is currently available concerning the endogenous concentration of the nucleoside in biological materials because of the technical difficulty of the measurement. The known methods with the use of enzymatic spectrophotometric (14, 15), radiochemical ( 16), fluorometric ( 17), microbiological ( 18) enzymatic isotope dilution (19), radioligand binding (20), and enzymatic fluorimetric (2 1) techniques are not sufficiently sensitive and are difficult to perform and are therefore not readily applicable to the routine analysis of large num409

bers of biological samples such as crude plasma and tissue extracts. A radioimmunoassay has been used for the sensitive and specific determination of numerous substances. This report describes a radioimmunoassay for the measurement of adenosine and includes a succinylation procedure which markedly enhances the assay sensitivity up to 0.25 pmol/tube. Application of the present method would be helpful for elucidating the physiological roles of adenosine. MATERIALS

AND METHODS

Reagents

Sources of reagents were as follows: human serum albumin (crystallized), Miles Laboratories, Elkhart, Indiana; 1-ethyl3 - (3 - dimethylaminopropyl)carbodiimide hydrochloride, Nakarai Chemicals Ltd., Kyoto, Japan; [ 2,5’,8-3H]adenosine and aqueous counting scintillant (ACS II), Amersham International Limited, Amersham, United Kingdom; complete Freund’s

0003-2697/82/060409-12$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

410

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adjuvant, Iatoron Laboratories, Tokyo, Jaadenosine deaminase, Boehringerw; Mannheim Yamanouchi k.k., Tokyo, Japan; ATP, ADP, AMP, adenosine, 2’deoxyadenosine, adenine, GTP, GDP, GMP, guanosine, guanine, IMP, inosine, hypoxanthine, UTP, UDP, UMP, uridine, uracil, TTP, TDP, TMP, thymidine, thymine, CTP, CDP, CMP, cytidine, cytosine, cyclic AMP, cyclic GMP, and cyclic CMP, Yamasa Shoyu Company, Ltd., Choshi, Chiba, Japan. Other reagents were of analytical grade from commercial sources. Synthesis

of 2’,3’-Disuccinyladenosine

Aqueous adenosine solution (6.7 g, 25 mmol in 500 ml) was added to 500 ml of a mixture of triethylamine and dioxane (1:9, v/v) which contained succinic anhydride (20 g, 200 mmol). The reaction mixture, after being kept at room temperature for 10 min under vigorous shaking, was concentrated in a rotary evaporator at 40°C under reduced pressure. The product was then dissolved in 1 liter of a hot solvent consisting of distilled water, ethanol, and dioxane (4:3:3, v/v). The crystal that appeared after cooling was dissolved again in the same solvent for purification. This recrystallization procedure was repeated three times to attain 100% purity, as revealed by high-pressure liquid chromatography. The yield of the pure product was 60%. Adenosine was quantitatively recovered on brief treatment of the product with 0.1 N NaOH. NMR study of this compound showed that the succinyl substitution was at the 2’- and 3’-0 positions. Preparation

of Immunogen

The immunogen was prepared by coupling the succinyladenosine thus prepared to human serum albumin. Succinyladenosine (200 mg) was mixed with 100 mg of human serum albumin and 100 mg of I-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride in 30 ml of 50 mtvr acetate buffer solution (pH 5.5). After incubation at 25°C

ET AL.

for 20 h in the dark, the mixture was dialyzed against 0.9% NaCl solution flowing at a rate of 400 ml/h for 48 h to separate the adenosine-protein conjugate from the free nucleoside derivative. The ultraviolet spectrum of the dialyzed conjugate had a maximum at 258 nm. From the difference between the extinction coefficient of succinyladenosine-albumin and that of unconjugated albumin, the conjugate was estimated to contain an average of 10 or 11 adenosine residues per albumin molecule. Preparation of Radioactive Disuccinyl[‘H]adenosine

[ 2,5’,8-3H]Adenosine (42 Ci/mmol in ethanol) was lOO-fold diluted with 50 mM acetate buffer solution (pH 6.5). To 1 ml of this diluted radioactive adenosine solution was added 1 ml of dioxane-triethylamine mixture (9:1, v/v) containing 40 mg of succinic anhydride. After being kept for 10 min at room temperature, the reaction mixture was diluted with 38 ml of 0.3 M imidazole buffer solution (pH 6.5). Electrophoresis on filter paper (Toyo Roshi No. 53) in 50 mM triethylamine bicarbonate buffer (pH 7.6) showed that the yield of the main product, 2’,3’-disuccinyl[3H]adenosine, was approximately 90%. Immunization

Schedule

Ten male rabbits of the Japanese white strain, weighing 1800-2200 g, were immunized with an emulsion composed of equal parts of complete Freund’s adjuvant and 0.9% NaCl solution containing succinyladenosine-albumin. They were given injections of 0.2, 0.2, and 0.2 mg of antigen in their backs at 1O-day intervals; 40 days later, the rabbits were immunized again the same way. All the animals were bled from the carotid artery 10 days after the last injection, when the antibody titer was expected to be maximal. The antisera were stored in small aliquots at -20°C.

ADENOSINE

Preparation of Dextran-Coated

immunoassay Procedure 1. Succinylation of adenosine. To 100 ~1 of a sample (a standard or test solution) was added 100 ~1 of the succinylating reagent. After being kept for 10 min at room temperature, the reaction mixture was diluted with 0.8 ml of 0.3 M imidazole buffer solution (pH 6.5). binding

TABLE

Charcoal

Dextran-coated charcoal, which had been used for cyclic nucleotide separation (22), was employed for adsorbing unbound adenosine. The dextran-coated charcoal was prepared by adding 500 mg of bovine serum albumin (Fraction No. V, Sigma Chemical Co., St. Louis, MO.), 75 mg of dextran (molecular weight 0.8-2.4 X lo’), and 500 mg of Norit Extra into 100 ml of distilled water. The mixture was stored in a cold room before use.

2. Antigen-antibody

411

RADIOIMMUNOASSAY

reaction.

To 100 ~1 of the succinylated sample was added 100 ~1 of 2’,3’-disuccinyl[3H]adenosine (18,000-25,000 cpm in an amount ranging between 0.5 and 1 pmol) and 100 ~1 of the diluted antiserum. The diluted antiserum was prepared by mixing the serum with 50 mM acetate buffer solution (pH 6.5) containing 0.3% bovine serum albumin and 10 mM MgCl,. T'he mixture was kept at 4°C for 4-24 h. 3. Separation of antiserum-bound adenosine from free adenosine. A cold solution

of dextran-coated charcoal (0.5 ml) was added to the above mixture cooled in an icecold water bath. Charcoal was then spun down, and 0.5 ml of the supernatant was counted for radioactivity in a liquid scintillation counter. High-Pressure Liquid Chromatography of Adenosine and Succinylated Derivatives

Adenosine and succinylated derivatives were chromotographed on a 0.4 X 15-cm spherical octadecylsilan silica (No. 3053,

BINDING

ACTIVITY

I

OF ADENOSINE

TO [‘HIADENOSINE

AND

ANTISERUM 2’,3’-

DISUCCINYL[~H]ADENOSINE

Binding activity, B/T (%) Antiserum No.

[‘HI-

Disuccinyl[‘H]adenosine

Dilution

Adenosine

1

1:3 I:600

3.9 0

14.2 30.0

2

1:3

2.6

I:600

0

63.7 43.1

3

I:3 1:600

2.2 0

82.1 52.4

4

1:3 I:600

6.0 0.7

71.3 43.5

5

1:3 I:600

3.2 0.6

67.7 38.8

6

I:3 I:600

2.5 0.5

67.8 30.5

Note. Incubation of labeled ligands with antiserum was carried out for 18 h in an ice-cold water bath. a Binding activity is expressed as B/T (o/o), that is, 100 times the ratio of the amount of radioactivity bound in the absence of adenosine (B) to that of total (bound + free) radioactivity (T).

Hitachi) column at room temperature using a mixture of CH3CN and 0.1 M KH,PO, at a flow rate of 1 ml/min with 100 kg/cm* pressure (a Hitachi high-pressure liquid chromatography apparatus, 635A). An ultraviolet detector at a wavelength of 260 nm equipped with a digital integrator (TakedaRiken, 2220A) was used for estimation of the yield of each product. Preparation of Biological Samples 1. Preparation of tissue extracts. Male Sprague-Dawley rats, weighing 200-250 g, were decapitated or anesthetized with pentobarbital after a fast of 18-20 h. Portions of the liver, kidney cortex, heart, lung, spleen, and pancreas were obtained and frozen immediately between stainless-steel tongs

412

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ET AL.

2’, 3’- Disuccinyladenosine

50%

a

I 100

10’ Nucleoslde N=leoslde

( pmol/

displacement

I

a

I

102

103

IO4

tube

)

FIG. 1. Effect of succinylation on assay sensitivity. The radioimmunoassay was carried out using adenosine antiserum at a I:600 dilution. Displacement is denoted by B/T (%), 100 times the ratio of the amount of radioactivity bound in the presence of each dose of the nucleoside (B) to that of total radioactivity (T).

cooled in liquid nitrogen. The frozen tissues were homogenized at 4°C in ice-cold 6% trichloroacetic acid; the homogenate was then centrifuged at 2540g for 15 min, and the supernatant was shaken three times with water-saturated ethyl ether to remove the acid. The extracted aqueous phase was directly assayed following the succinylation. 2. Preparation of plasma samples. The blood collected in a heparinized tube was mixed with an aqueous solution of MgC& and dipyridamole to make their final concentrations 10 mM and O.Ol%, respectively. Immediately after mixing, the blood was centrifuged at 2540g for 5 min at 4°C. The plasma obtained in this manner was directly succinylated and submitted to immunoassay. Adenosine Deaminase Treatment of Biological Materials

Samples (tissue extracts, plasma, or standard solution) were incubated for 1 h at

45°C in 400 ~1 of phosphate buffer solution (50 mM, pH 7.4) containing adenosine deaminase from calf intestine (40 mu/tube). Afterward adenosine in the incubation medium was directly succinylated and determined. RESULTS Adenosine Antibody

All rabbits immunized with the succinyladenosine-human serum albumin conjugate had serum titers of adenosine antibody. From 6 of the 10 rabbits immunized, hightiter antisera were obtained; these antisera were capable of binding more than 30% of 2’,3’-disuccinyl[3H]adenosine (0.7 pmol) at a dilution of 1:600 after an 18-h incubation. The binding activity of each antiserum to nonsuccinylated[3H]adenosine (0.7 pmol) was much lower than that to disuccinyl[3H]adenosine, even at a minimal dilution (1:3) (Table 1). Figure 1 shows the

ADENOSINE

displacement curves of disuccinyl[ 3H]adenosine with disuccinyladenosine and unmodified adenosine. The concentrations of each nucleoside that caused 50% displacement of disuccinyl[ 3H]adenosine were 2 pmol for 2’,3’-disuccinyladenosine and 1000 pmol for adenosine. It is evident that conversion of adenosine in samples into 2’,3’-disuccinyladenosine before binding to the antibody made the assay 500-fold more sensitive. The specificities of the antisera are shown in Table 2. Among the structurally related compounds, only 2’-deoxyadenosine treated with the succinylating reagent was cross-reactive; the relative strength of binding of this compound to the adenosine antibody was about 1.3% of that of adenosine. Accordingly, this nucleoside should be removed from the sample or decomposed prior to the assay. We adopted a method of selective decomposition by heating the sample with 0.1 N HCl at 50°C for 30 min; with this TABLE CROSS-REACTIVITY

Compound

tested

S-Adenosineb S-Deoxyadenosine S-ATP S-ADP S-AMP S-Adenine S-GTP S-GDP S-GMP S-Guanosine S-Guanine S-IMP S-Inosine S-Hypoxanthine S-UTP S-UDP

OF VARIOUS

413

RADIOIMMUNOASSAY

procedure, deoxyadenosine was completely decomposed into adenine and 2-deoxyribose, which never interfered with adenosine binding to the antibody, while the amount of adenosine remained unaltered (Fig. 2). Application of this decomposing procedure to biological samples prepared from various rat tissues caused no significant change in their adenosine values; e.g., 6.2 and 6.0 (heart), 3.0 and 3.2 (kidney cortex), 1.1 and 1.2 (lung), and 2.2 and 2.0 (plasma) were the amounts (pmol) per assay tube before and after treatment, respectively. Thus, the biological materials appear to contain little, if any, 2’-deoxyadenosine. A 12,000-fold greater concentration of succinyl AMP and a 21,000-fold greater concentration of succinyl 3’,5’-cyclic AMP were required to produce displacement of radioactive ligand to extents comparative to that caused by a concentration of 2’,3’-disuccinyladenosine. As for all other purine and pyrimidine compounds tested, the cross-reactivity was neg2

COMPOUNDS

WITH ADENOSINE

ANTISERUM

Relative binding affinity’

Compound tested

Relative binding affinity”

I00.000 1.320 to.002 -co.002 0.008 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002

S-UMP S-Uridine S-Uracil S-TTP S-TDP S-TMP S-Thymidine S-Thymine S-CTP S-CDP S-CMP S-Cytidine S-Cytosine S-Cyclic AMP S-Cyclic GMP S-Cyclic CMP

<0.002 <0.002 <0.002 co.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 10.002 <0.002 10.002 0.005 to.002 10.002

NOW. The radioimmunoassay was performed as described under Materials and Methods by incubating reaction mixture for 18 h in an ice-cold water bath using the adenosine antiserum at a I:600 dilution. ’ Relative binding affinity was calculated as [S-adenosine]/[compound tested] X 100, where [S-adenosine] [compound tested] are the concentrations required to cause a 50% displacement of ‘H-ligand binding. b The prefix S indicates that the compounds have been treated with the succinylating reagent.

the and

414

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ET AL.

0 0

5

10

20

FIG. 2. Effect of HCI deoxyadenosine (50 pM, by radioimmunoassay.

(min

60

)

treatment on adenosine and 2’-deoxyadenosine. Adenosine (1 pM, 0) and 2’A) were subjected to HCI treatment, as described in the text, and determined

ligible (<0.0017%). Thus, these adenosinerelated compounds cause no significant interference owing to the high specificity of the antiserum. Quantitative

45

30 lime

Succinylation

of Adenosine

As the assay sensitivity was markedly improved by succinylation of adenosine in test fluids, the experimental conditions required for quantitative succinylation were investigated in detail. We used dioxane as a convenient solvent for succinic anhydride, which has to be kept in a nonaqueous phase before its addition to test solutions because of its miscibility with water and its failure to interfere with the antigen-antibody binding reaction. The amount of the reagents required for quantitative succinylation of adenosine was determined as follows: Adenosine at a high concentration (5 pmol/ 100 ~1) was mixed with varying amounts of succinic anhydride in 100 ~1 of a dioxane-triethylamine mixture. The reaction mixtures were analyzed for the yield of succinylated adenosine by high-pressure liquid chroma-

tography. The representative elution profiles are shown in Fig. 3. A combination of 2 to 4 mg of succinic anhydride and 2.5 to 10 ~1 of triethylamine was effective in converting adenosine to 2’,3’-disuccinyladenosine with a yield of 86 to 88%. The succinylation reaction occurred spontaneously in only a few seconds, and the value obtained by radioimmunoassay was not altered by prolongation of the reaction time up to 14 h. No interference was caused by the coexistence of plasma or various tissue extracts (Table 3). Time for Equilibration Antibody Reaction

of Antigen-

The antigen-antibody reaction reached 50 and 100% equilibrium within 2 and 8 h, respectively. The slope of the standard curve was steeper after 8 h of incubation than after 4 h or less (Fig. 4). Validity

of the Assay

I. Dilution test. As shown in Fig. 5, the value of adenosine obtained by the present

ADENOSINE

: 9:

415

RADIOIMMUNOASSAY

1Iv

@

9 9

0.5.

O-

I 5

0

I 15

I 10

FIG. 3. High-pressure liquid chromatography of adenosine and succinylated derivatives. One microliters of adenosine solution was mixed with 100 ~1 of the reagent consisting of 1 mg of anhydride, 2.5 ~1 of triethylamine, and 97.5 ~1 of dioxane (A) or 4 mg of succinic anhydride, triethylamine, and 90 ~1 of dioxane (B). Each peak was identified by its ultraviolet spectrum, phoretic mobility, and retention time on high-pressure liquid chromatography: I, adenosine; II monosuccinyladenosine; IV, 2’,3’-disuccinyladenosine; V, trisuccinyladenosine.

0.25

0.5

1

2 S-Adenosinc

FIG. 4. Effect of incubation

4

6 ( pmol/tuba

time on standard

16 1

curves.

32

64

hundred succinic IO ~1 of electroand III,

416

= 0 e a 2 g20-

SAT0

0.5

15

1

ET AL.

2 ( mg ) 10

@

@ r

6 4 1 51

lo

d d 0

1.5

1

0.5

0

2

0.25

0.5

Amount

FIG. 5. Determination of rat liver (A), heart

of adenosine (B), lung (C),

TABLE SUCCINYLATION BIOLOGICAL

Sample Water Rat liver extract Rat heart extract Rat lung extract Rat plasma Human plasma

OF ADENOSINE

IN

SAMPLES

Amount of tissue or plasma in 100 pl 0 IO mg I4 mg

8 mg 99 pl 99 ccl

of

Succinylation yield (%) 86.5 87.4 88.1 86.1 86.0 87.2

Note. Plasma and extracts of the tissue were prepared as described under Materials and Methods. [“HIAdenosine (22,000 cpm, 1 pmol) was added to 100 pl of the tissue extract, plasma, or water and mixed with the succinylating reagent, which consisted of 4 mg of succinic anhydride, 10 pl of triethylamine, and 90 ~1 of dioxane. After IO min, the mixture was diluted with water and submitted to paper electrophoresis together with authentic adenosine and 2,‘,3’-disuccinyladenosine. Spots of succinylated derivatives were extracted with water and counted for triiium to estimate the conversion rate of [‘Hladenosine to disuccinyl[‘H]adenosine.

)

samples

in varying sample sizes. Adenosine and plasma (D) were determined

3

1

0.75 ( pl

( mg )

contents in increasing by radioimmunoassay. ,

amounts

assay method was proportional to the amounts of the biological materials in the test sample, thus excluding the possibility that these materials, tissue and plasma, contained any substance which interfered with the assay. 2. Recovery test. Adenosine added to plasma and heart extract was quantitatively recovered (Table 4) again indicating that there were no inhibitory or interfering substances in these biological materials. 3. Digestion test. To determine whether the immunoreactive substance was adenosine, biological samples were incubated with adenosine deaminase before the assay; no immunoreactive substance was detected after the incubation, indicating that the immunoreactive substance in the samples, if such a substance existed, was essentially an adenosine deaminase-susceptible compound, adenosine or 2’-deoxyadenosine (Table 5). 4. Reproducibility of the assay. The between-assay reproducibility was studied at three levels of adenosine concentration, as

ADENOSINE TABLE RECOVERY

OF ADENOSINE AND

TABLE

4 ADDED

HEART

417

RADIOIMMUNOASSAY

TO RAT

PLASMA

BETWEEN-ASSAY

6

REPRODUCIBILITY’

EXTRACT Adenosine

Adenosine Biological material

Recovery Added

Found

0 0.9 I.8

2.5 + 0.1 3.6 t 0.1 4.2 + 0.1

3.4 4.3

105.9 91.1

0 I.7 3.4

6.3 + 0.2 7.8 k 0.1 9.8 f 0.6

8.0 9.7

91.5 101.0

Plasma

Heart

extract

Now. Values

Calculated

shown in Table 6. The coefficient of variance at the low or middle concentration of adenosine was smaller than that at the highest concentration studied, probably because the standard curve was more linear at the low or middle concentration than at the highest concentration. The intraassay coefficient of variance for six aliquots from the same tissue extract analyzed in a single assay was 3.8%. Both the between-assay and the intraassay reproducibilities seem satisfactory.

1. Tissue content of adenosine. The tissue contents of adenosine ranged from a few

EFFECT

OF ADENOSINE

LEVELS

IN RAT

Mean (pmol/assay tube) Standard deviation Coefficient of variance (90)

5

DEAMINASE ON ADENOSINE TISSUES AND FLUIDS

deaminase tEiltltle”t After

Adenosine solution Heart extract’ Pancreas extract0 Kidney cortex extract’ Plasmab Urine’

20.0 15.8 6.1 16.1 6.0 8.5

0 0.32 0 0.25 0 0

High

I .05 0.07 6.1

4.05 0.24 5.9

16.35 2.16 13.2

TABLE CONCENTRATION TISSUES

Disappearance (%) loo.0 98.0 100.0 98.5 100.0 100.0

Now. Values are given in pmol per tube. *Tissues were obtained after decapitation. b Blood was collected by decapitation. and plasma WBS directly treated with the enzyme. ‘Urine was IO-fold diluted and treated with the enzyme. See Materials and Methods for details.

by two mvestigators.

I

OF ADENOSINE AND

IN VARIOUS

BIOLOGICAL

FLUIDS

Adenosine (nmol/g Rat tissue (3)

Decapitation”

Heart Lung Liver Spleen Pancreas Kidney cortex

16.43 8.49 9.00 4.67 18.70 16.57

Biological

Before

Middle

nanomoles to about 20 nmol/g wet wt of the tissues tested (Table 7). Our value for rat heart adenosine is a few times higher than that reported by Berne et al. (4) but about one-half of that reported by Namm and Leader (19). Table 7 further shows that decapitation of rats resulted in adenosine levels in heart, pancreas, and kidney cortex that were significantly higher than the levels observed after pentobarbital anesthesia.

Adenosinc

Sample

LOW

‘The number of assays was six performed b Sample was rat heart CX~TBC~.

of Adenosine

Concentration

TABLE

in samples*

(%)

are meam + SD (n = 3) in pmol per tube.

Biological

concentration

(pmol)

fluid

Rat plasma’ (16) Rat urine (8) Human plasma” (5) Human urine (3) Canine plasmad (3)

+ + + f + +

wet wt of tissue) Anesthesia’

1.94’ 1.72 1.01 0.04 O.OSb 0.94’

10.62 7.58 1.79 3.67 13.27 7.94

r 0.02 2 1.16 31 1.16 k 0.43 k 0.36 ? 0.78

Adenosine (nmol/ml) 7.55 16.63 0.29 1.56 0.15

f + k k ?I

0.51 2.01 0.08 0.57 0.03

NOW. Values represent means + SEM. The number of determinations is given in parentheses. ’ A portion of tissues was excised from each animal immediately after decapitation (decapitation) or under pentobarbital anesthesia (anesthesia). ’ Significantly higher than that obtained with pentobarbital. ’ Blood was obtained by decapitation. * Blood was drawn from the cubitus vein.

418

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ET AL.

I

I

1

15

30

60

Time

(min

)

0

I

I

1

15

30

60

Time

(min)

FIG. 6. Spontaneous decrease in concentration of adenosine in rat plasma and blood and its inhibition. Blood was collected from the carotid arteries of anesthetized rats. A portion of the heparinized blood was immediately centrifuged, and adenosine was added to the resultant plasma at a final concentration of 1000 pmol/ml, which was stored at O”C, as shown on the abscissa of Panel A. Residual whole blood, to which adenosine had been added at a final concentration of 1000 pmol/ml, was stored at 0°C for the times indicated on the abscissa of Panel B; at each time, a fraction was withdrawn for separation of the plasma. Plasma concentration of adenosine is plotted against time of storage: 0, without any addition; A, with IO mM MnQ; n , with 0.01% dipyridamole; *, with 10 mM MnC12 and 0.01% dipyridamole.

2. Plasma concentration ofadenosine. As is shown in Fig. 6A, the plasma concentration of adenosine rapidly decreased during maintenance of the plasma in an ice-cold water bath, probably because of degradation by adenosine deaminase originating from blood cells. The breakdown of adenosine was completely inhibited by addition of MnCl, at a final concentration of 10 mM. The plasma concentration of adenosine was rapidly lowered, even in the presence of 10 mM MgC&, however, when the sample was stored as the whole blood system (Fig. 6B). Thus, the adenosine value estimated in separate plasma samples appears not to reflect the true value in the original plasma of the circulating blood because of its decrease during centrifugation of heparinized blood. The decrease of the adenosine concentration in the plasma fraction during storage of the whole blood may be due to its uptake into blood cells, since the decrease was prevented by

dipyridamole, a potent uptake inhibitor (Fig. 6B). Thus, addition of MnC12 (10 mM) in combination with dipyridamole (0.01%) completely inhibited the decrease in the plasma concentration of adenosine during 60-min maintenance of whole blood. Table 7 shows the plasma concentration of adenosine determined in this manner; the blood was rapidly mixed with MnC12 and dipyridamole immediately after withdrawal. Rubio et al. reported that the concentration of adenosine in dog coronary sinus blood collected during the reactive hyperemic period was 130 pmol/ml of blood (23), and Mills et al. reported that the adenosine concentration of normal human plasma is 0.31 PM (2). DlSCUSS(ON

The present method for the determination of adenosine in biological materials seems superior to previously reported methods ( 14-

ADENOSINE

419

RADIOIMMUNOASSAY

21) because of its high sensitivity and specificity. The high sensitivity of the radioimmunoassay permits the measurement of adenosine in less than 1 mg wet wt most tissues. The marked specificity of the antiserum eliminates the need for additional purification procedures such as chromatography (20), and hence allows a rapid and precise analysis of large numbers of biological samples. It is generally accepted that the affinity of competitors for proteins is one of the most important factors in determining the sensitivity of a competitive binding assay. In the present method, the succinylating reagent converts adenosine into 2’,3’-disuccinyladenosine, which has a higher affinity for the antibody that has been produced recognizing a moiety of immunogens containing disuccinyl linkages. Actually, the sensitivity of the assay was 500-fold higher when adenosine in the samples was succinylated prior to the competitive binding reaction (Fig. 1). This effect of succinylation on assay sensitivity is in good agreement with the original observation of Steiner et al. (24) that 2’-O-acylated cyclic nucleotides have higher affinities for their antibodies than the nonsubstituted cyclic nucleotides. It should be noted here, however, that succinylation resulted in a much greater increase in sensitivity for adenosine (about 500-fold) than for cyclic nucleotides (about loo-fold) (22, 25). This difference appears to be based on the differences in the degree of succinylation; two hydroxyl groups are succinylated on a molecule of adenosine, while only one is succinylated on the cyclic nucleotide molecule. Although the succinylation of adenosine may produce several derivatives, we established that succinylation selectively occurs at the 2’- and 3’0 positions. Such selective succinylation would have contributed to the increase in sensitivity of the assay. The validity of the radioimmunoassay technique for measuring adenosine in tissues and biological fluids was demonstrated by the dilution test, recovery test, and adenosine deaminase treatment. In the case of aden-

osine levels in heart extracts, our data are similar to those obtained by other investigators with an enzymatic spectrophotometric technique (14, 15) and an enzymatic isotope dilution method ( 19). One advantage of the present method is that the adenosine present in plasma can be assayed directly without prior deproteinization, owing to the stable binding of antigen-antibody complexes in the imidazole buffer solution (O.l0.5 M), which was previously reported to be useful in assays of cyclic nucleotides by Honma et al. (22). The plasma concentration of adenosine progressively decreased during short-term storage of whole blood, even in an ice-cold water bath (Fig. 6). The combined addition of MnC12 and dipyridamole to the blood sample to prevent adenosine from undergoing enzymatic degradation and uptake by blood cells made it possible to obtain reliable values for adenosine in circulating blood. In view of important physiological roles of adenosine as an extracellular agonist for its specific membrane receptors in a variety of cell types, the plasma concentration of adenosine assayable by the present technique would serve as a good index of some cellular functions in the whole body. The increase in the adenosine content of several tissues that occurred after decapitation of the tissue-donor animal might be a reflection of ischemia or hypoxia, which should cause generation of adenosine from adenine nucleotides. Thus, the present method, which is sensitive, simple, and reproducible, is expected to contribute to further elucidation of the role of adenosine in various biological and biochemical systems. ACKNOWLEDGMENTS The authors would like to thank Mr. M. Morozumi, Dr. S. Shibuya, and Mr. M. Kumagai for their help in the synthesis of 2’,3’-disuccinyladenosine.

REFERENCES 1. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., and Meuwissen, H. J. (1972) Lancer 2, 10671069.

420

SAT0

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