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Clinical Chemistry 46: 537-542, 2000;
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(Clinical Chemistry. 2000;46:537-542.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Simple and Sensitive Binding Assay for Measurement of Adenosine Using Reduced S-Adenosylhomocysteine Hydrolase

Doris Kloor1,a, Kozo Yao2, Ursula Delabar1 and Hartmut Osswald1

1 Department of Pharmacology, Faculty of Medicine, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany.

2 Department of Pharmacology, Kyowa Hakko Kogyo Co. Ltd, Mishima 411-8731, Japan.
a Author for correspondence. Fax 49-07071-294942; e-mail doris.kloor{at}uni-tuebingen.de


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [3H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form.

Methods: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid).

Results: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 ± 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC.

Conclusions: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adenosine, produced from either extracellular AMP by ecto-nucleotidase (1) or intracellular AMP (2), interacts in physiological processes with hormones and neurotransmitters (3). Another source for adenosine generation is the pathway leading from S-adenosylmethionine via S-adenosylhomocysteine (SAH) to adenosine. Adenosine thus is an obligatory product of the S-adenosylmethionine-dependent methylation reaction. The key enzyme in this pathway directly leading to the formation of adenosine is SAH hydrolase (EC 3.3.1.1), which was first described by de la Haba and Cantoni (4) in rat liver. The reaction catalyzed by this enzyme is reversible, although the thermodynamic equilibrium favors the SAH synthesis from adenosine and homocysteine (4). Adenosine has been suggested as important in the control of coronary blood flow (5)(6), cardiac arrhythmias (7), the inhibition of adrenergic activity at pre- and postsynaptic sites (8), and in the regulation of renal function (9)(10). This nucleoside modulates several physiological effects by stimulating specific cell surface receptors (11)(12). Because the extracellular concentration of adenosine is ~10-8 to 10-9 mol/L (13) and because the intracellular free adenosine concentration is estimated as ~10-8 mol/L (14), the concentration of adenosine is at or below the limit of detection of most analytical methods. Most of the described methods require either expensive equipment or prepurification or succinylation of adenosine-containing samples. HPLC procedures either use prior purification or lack adequate sensitivity and require large amounts of samples (15). In addition tissue, plasma, urine, and cerebrospinal fluid also contain adenine nucleotides, and a specific method is necessary for reliable estimations.

Here we describe a sensitive and specific adenosine-binding protein assay (ABPA) for detection of adenosine in samples that can be directly applied to deproteinized specimens.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
The following materials were purchased from the sources indicated: [2,8,5'-3H]adenosine (2.3 TBq/mmol) from NEN; adenosine, AMP, ADP, ATP, cAMP, adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase from Boehringer Mannheim Germany; SAH, adenine, 2'-deoxyadenosine, L-homocysteine, diadenosine diphosphate, N6-methyladenosine, and cGMP from Sigma; and 0.45 µm nitrocellulose filters from Schleicher and Schuell.

All other chemicals were of analytical grade and obtained from Merck.

enzyme purification
SAH hydrolase was purified from bovine kidney with chromatographic techniques as described previously (16). The purified enzyme was frozen at -20 °C until use.

protein determination
Protein concentrations were determined according to the method of Bradford (17), using bovine serum albumin as the calibrator.

enzyme activity of sah hydrolase
The SAH hydrolysis activity was assayed in a total volume of 1 mL at 20 °C. The reaction mixture contained 80 µmol/L SAH, 2 kU/L adenosine deaminase, 0.8 kU/L nucleoside phosphorylase, 0.8 kU/L xanthine oxidase, and 50 mmol/L potassium phosphate, pH 7.0.

The reaction was started by the addition of 20 mg/L SAH hydrolase. The uric acid formed was measured photometrically at 292 nm.

assay method
The principle of this assay is based on the ability of the enzyme SAH hydrolase to bind adenosine. The competitive ABPA for adenosine uses SAH hydrolase in its reduced, enzymatically inactive form as the binding protein. As shown previously, enzymatically active SAH hydrolase from bovine kidney binds [3H]adenosine with a high-affinity dissociation constant of 6.8 nmol/L (16). The reduction of SAH hydrolase leads to an enzymatically inactive enzyme that retains its ability to bind adenosine with high affinity (18).

preparation of reduced (nadh)-sah hydrolase
The tightly bound NAD+ of the SAH hydrolase was removed by incubation of the native enzyme (100 µg of protein) with 100 µL of incubation buffer (150 mmol/L KCl, 80 mmol/L ATP, and 80 mmol/L MgCl2). After incubation for 120 min at 37 °C, the enzyme activity of the SAH hydrolase in this mixture was measured as described above; the enzyme solution was then dialyzed against 15 mmol/L Tris–20 mmol/L HEPES, pH 7.0. Without NAD+, the enzyme is completely inactive and loses its binding affinity to adenosine. The reconstitution of the enzyme with 1 mol/L NADH in 15 mmol/L Tris–20 mmol/L HEPES, pH 7.0, produces SAH hydrolase in its reduced (NADH-SAH hydrolase) form (19). The enzyme activity of this reduced enzyme was tested to confirm the inactivation of the SAH hydrolase. This reduced and enzymatically inactive SAH hydrolase was stored in 100-µL aliquots at -20 °C. These aliquots contained a protein concentration of 600–1000 mg/L. In this form and at the temperature indicated above, the reduced enzyme is stable for at least 2 months.

quantification of adenosine by binding assay procedure
Displacement of [3H]adenosine was performed in a final assay volume of 300 µL of 20 mmol/L Tris–40 mmol/L HEPES, pH 7.4, with a concentration of SAH hydrolase of 3 mg/L (1 µg/300 µL assay volume), a fixed concentration of [3H]adenosine (3 nmol/L; 1 pmol/300 µL assay volume), and various concentrations (1, 10, 30, 100, 300, and 1000 nmol/L) of unlabeled adenosine. The sample volume in the assay was 50 µL. The maximum sample volume used in the assay was 150 µL; the minimum sample volume was 20 µL. After incubation for 14 h at 20 °C, the assay mixture was filtered through nitrocellulose filters. The filters were washed with 4 mL of 20 mmol/L Tris–40 mmol/L HEPES, pH 7.4. The radioactivity adsorbed on the filters was determined by liquid scintillation counting with Ultima Gold® (Packard) as scintillation fluid in a model 2550TR liquid scintillation analyzer (Packard).

To construct a calibration curve, we plotted the log values of the concentration of adenosine against the [3H]adenosine bound (as a percentage) and fitted the curve with a four-parameter logistic equation. The adenosine values between 10-9 and 10-6 mol/L from the resulting curve were then used to calculate the adenosine values of unknown samples on the basis of the counts per minute observed.

urine samples
Urine samples were collected in tubes containing 10 g/L sulfosalicylic acid. Deproteinated undiluted samples may be stored frozen at -80 °C without loss of adenosine for 3 month. Before measurement, 500 µL of each sample was neutralized to a pH between 7.0 and 7.8 with 50 µL of 2.5 mol/L ammonium acetate, pH 8.7.

tissue samples
Rat kidneys shock-frozen to the temperature of liquid nitrogen were powdered under liquid nitrogen and transferred into a preweighed vial containing 6 mL of precooled 0.6 mol/L perchloric acid. After centrifugation at 12 000g for 30 min at 4 °C, the supernatant was collected and 500 µL of the supernatant was adjusted to a pH between 7.0 and 7.8 by the addition of 50 µL of 2 mol/L potassium carbonate, pH 9.5.

For comparison, all samples presented here were also analyzed for adenosine by HPLC (15).

calculation and statistics
The Student t-test for unpaired values was used to determine the levels of significance. The data were analyzed using linear regression analysis. The run test was used to determine the goodness of fit of data to a given curve.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
characteristics of adenosine binding to nadh-sah hydrolase
Saturation binding experiments (Fig. 1 ) were performed in the presence of 0.5–300 nmol/L [3H]adenosine. Nonspecific binding increased linearly with increasing [3H]adenosine concentrations and represented ~3–5% of total binding of adenosine (16). Adenosine binds to the reduced enzyme compared with the active enzyme with an affinity (Kd) of 32 ± 2 nmol/L vs 12.4 ± 0.8 nmol/L. The Bmax of the NADH-SAH hydrolase was enhanced compared with the native enzyme by a factor of 3, from 238 ± 3.2 pmol/mg to 848 ± 19 pmol/mg protein. At 20 °C, the binding of 3 nmol/L [3H]adenosine to NADH-SAH hydrolase reached equilibrium after 8 h, whereas at 4 °C, the binding of 3 nmol/L [3H]adenosine reached equilibrium after 22 h (Fig. 2 ).



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  		Figure 1. Specific binding of [3H]adenosine to NAD+-SAH (•) and NADH-SAH ({blacksquare}) hydrolase.

SAH hydrolase was incubated with increasing concentrations of [3H]adenosine (0.5–300 nmol/L).



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  		Figure 2. Association kinetics of specific [3H]adenosine binding to NADH-SAH hydrolase at 20 °C (•) and 4 °C ({blacksquare}).

NADH-SAH hydrolase was incubated with 3 nmol/L [3H]adenosine for various lengths of time.

calibration curve
A representative calibration curve for the binding of adenosine is shown in Fig. 3 . Because the human urine and rat kidney tissue samples contained between 0.5 and 50 µmol/L adenosine, the samples were diluted in 20 mmol/L Tris–40 mmol/L HEPES, pH 7.4.



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  		Figure 3. Typical calibration curve prepared with [3H]adenosine and with unlabeled adenosine.

The adenosine concentrations used to determined the unknown adenosine concentration in the samples were 1, 10, 30, 100, 300, and 1000 nmol/L. The calibration curve was constructed by fitting the concentration vs percentage bound data with a four-parameter logistic function.

interference of adenosine analogs
The interference of adenosine analogs with the binding of [3H]adenosine to NADH-SAH hydrolase was analyzed by displacement experiments. The IC50 values of these compounds are summarized in Table 1 . Of the endogenous substances, 2'-deoxyadenosine was the only compound that affected the assay when present in samples in a concentration >100 nmol/L.


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  		Table 1. IC50 values of 3 nmol/L [3H]adenosine displacement using NAD+- and NADH-SAH hydrolase.

Because under normoxic conditions, the concentrations of the adenine nucleotides in the rat kidney are 3 mmol/L ATP, 0.4 mmol/L ADP, and 0.2 mmol/L AMP, and because the intracellular concentrations of SAH and cAMP are ~1 nmol/L and 0.1 µmol/L, respectively, displacement of the bound [3H]adenosine from the NADH-SAH hydrolase under the assay conditions is unlikely. In addition, N6-methyladenosine, which often is used as internal standard for HPLC, shows a high affinity to NADH-SAH hydrolase (IC50 = 730 nmol/L).

linearity and recovery
Table 2 shows the results for analysis of dilutions of a urine sample with an adenosine concentration of 311 nmol/L, as determined by HPLC and ABPA and then diluted in 20 mmol/L Tris–40 mmol/L HEPES, pH 7.4. Linear regression of the observed adenosine (y) vs the calculated expected adenosine (x) gave the following equation: y = 0.96x - 0.5 (r = 0.999; Sy|x = 0.963).


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  		Table 2. Dilution linearity.

In the recovery test with rat and human urine and rat kidney tissue samples (Table 3 ), recovery of the added adenosine was 96.4–107%. These results demonstrate that there were no inhibitory or interfering substances in the urine and kidney tissue.


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  		Table 3. Recovery of adenosine added to rat urine (samples 1 and 2), human urine (sample 3), and tissue (sample 4).

precision and comparison of methods
The intra- and interassay imprecision of the method (as CVs) is shown in Table 4 . The intraassay CV was determined by analyzing three samples of rat urine containing 32.1, 47.4, and 97.0 nmol/L adenosine in 5 parallel determinations and two samples of rat kidney tissue containing 4.4 and 20.7 µmol/L in 12 parallel determinations. The interassay CV was determined by measuring each urine and tissue sample on 5 different days. The intra- and interassay CVs were 1.2–3.9% and 3.0–7.8%, respectively.


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  		Table 4. Intra- and interassay imprecision of the method.

Adenosine in 18 rat urine, 51 human urine, and 47 rat kidney tissue samples was determined by ABPA and HPLC according to the method of Delabar et al. (15). A comparison of the results obtained with the method presented here and those of the HPLC method indicated good agreement between the methods (Figs. 4 and 5). The correlation coefficient between the values obtained by these two methods was 0.901 (Sy|x = 1.12) for urine and 0.966 (Sy|x = 0.92) for tissue, respectively.



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  		Figure 4. Comparison of adenosine measurements in urine samples by ABPA (x axis) vs HPLC (y axis).

(A), linear regression analysis: y = 1.02x - 0.44 (n = 51; r = 0.901; Sy|x = 1.12). (B), Bland-Altman plot (22), which shows the difference in adenosine results between the two methods as a function of their mean value.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of the present study was to establish a simple and sensitive binding assay for adenosine measurements in small samples without extensive purification procedures. Most methods for determining adenosine in biological samples are time-consuming, or need sample purification (20) or succinylation (21).

We used SAH hydrolase in its NADH form as a specific adenosine-binding protein. This enzymatically inactive protein retains its adenosine-binding capacity and has the advantage that no formation of SAH from adenosine and homocysteine, or hydrolysis of SAH can take place in the incubation mixture. In addition, the interference of endogenous adenine and adenine nucleotides in the binding of adenosine to NADH-SAH hydrolase is no longer present. Furthermore, NADH-SAH hydrolase has a threefold higher binding capacity for adenosine. Thus, only 1 µg of protein per assay volume (300 µL) is required to detect 10 nmol/L adenosine.

The binding assay with the NADH-SAH hydrolase and [3H]adenosine of high specific activity is sensitive enough to detect adenosine in samples with small volumes. Therefore, it is possible to detect physiological changes in adenosine concentrations. The precision of the method was satisfactory (CV <3.9–7.8%), and it has sufficient analytical range. The adenosine results obtained correlated well with HPLC results. The Bland-Altman plot (Fig. 4BUp ) indicated that the method presented here has a tendency to give lower adenosine values in urine samples in the higher concentration range (y = -0151x + 0.656), whereas the tissue samples showed an even distribution in the Bland-Altman plot (Fig. 5B ; y = 0.058x - 1.143). The ABPA procedure is extremely simple and does not require any complicated purifications when the samples are deproteinized. The method is an attractive alternative to HPLC analysis in both routine and research laboratories. This analytical method may help clinical researchers investigate the physiological roles and therapeutic potencies of adenosine and several adenosine derivatives for treating diseases in which adenosine metabolism is disturbed and, therefore, organ function is impaired.



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  		Figure 5. Comparison of adenosine measurements in tissue samples by ABPA (x axis) vs HPLC (y axis).

(A), linear regression analysis: y = 0.9125x + 1.70 (n = 47; r = 0.966; Sy|x = 0.912). (B), Bland-Altman plot, which shows the difference in adenosine results between the two methods as a function of their mean value.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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