Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase.
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
The following materials were purchased from the sources indicated: [2,8,5'[sup.3]H]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, [N.sup.6]-methyladenosine, and cGMP from Sigma; and 0.45 [micro]m nitrocellulose filters from Schleicher and Schnell.
All other chemicals were of analytical grade and obtained from Merck.
SAH hydrolase was purified from bovine kidney with chromatographic techniques as described previously (16). The purified enzyme was frozen at -20[degrees]C until use.
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[degrees]C. The reaction mixture contained 80 [micro]mol/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.
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 [[sup.3]H]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.sup.+] of the SAH hydrolase was removed by incubation of the native enzyme (100 [micro]g of protein) with 100 [micro]L of incubation buffer (150 mmol/L KCI, 80 mmol/L ATP, and 80 mmol/L [MgCl.sub.2]). After incubation for 120 min at 37[degrees]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.sup.+], 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-[micro]L aliquots at -20[degrees]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 [[sup.3]H]adenosine was performed in a final assay volume of 300 [micro]L of 20 mmol/L Tris-40 mmol/L HEPES, pH 7.4, with a concentration of SAH hydrolase of 3 mg/L (1 [micro]g/300 [micro]L assay volume), a fixed concentration of [[sup.3]H]adenosine (3 nmol/L; 1 pmol/300 [micro]L 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 [micro]L. The maximum sample volume used in the assay was 150 /,L; the minimum sample volume was 20 [micro]L. After incubation for 14 h at 20[degrees]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[R] 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 [[sup.3]H]adenosine bound (as a percentage) and fitted the curve with a four-parameter logistic equation. The adenosine values between [10.sup.-9] and [10.sup.-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 were collected in tubes containing 10 g/L sulfosalicylic acid. Deproteinated undiluted samples may be stored frozen at -80[degrees]C without loss of adenosine for 3 month. Before measurement, 500 [micro]L of each sample was neutralized to a pH between 7.0 and 7.8 with 50 [micro]L of 2.5 mol/L ammonium acetate, pH 8.7.
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 pre-cooled 0.6 mol/L perchloric acid. After centrifugation at 12 000g for 30 min at 4[degrees]C, the supernatant was collected and 500 [micro]L of the supernatant was adjusted to a pH between 7.0 and 7.8 by the addition of 50 [micro]L 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 Mest 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.
CHARACTERISTICS OF ADENOSINE BINDING TO NADH-SAH HYDROLASE
Saturation binding experiments (Fig. 1) were performed in the presence of 0.5-300 nmol/L [[sup.3]H]adenosine. Non-specific binding increased linearly with increasing [[sup.3]H]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 ([K.sub.d]) of 32 [+ or -] 2 nmol/L vs 12.4 [+ or -] 0.8 nmol/L. The [B.sub.max], of the NADH-SAH hydrolase was enhanced compared with the native enzyme by a factor of 3, from 238 [+ or -] 3.2 pmol/mg to 848 [+ or -] 19 pmol/mg protein. At 20[degrees]C, the binding of 3 nmol/L [[sup.3]H]adenosine to NADH-SAH hydrolase reached equilibrium after 8 h, whereas at 4[degrees]C, the binding of 3 nmol/L [[sup.3]H]adenosine reached equilibrium after 22 h (Fig. 2).
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 [micro]mol/L adenosine, the samples were diluted in 20 mmol/L Tris-40 mmol/L HEPES, pH 7.4.
INTERFERENCE OF ADENOSINE ANALOGS
The interference of adenosine analogs with the binding of [[sup.3]H]adenosine to NADH-SAH hydrolase was analyzed by displacement experiments. The [IC.sub.50] 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.
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 /,mol/L, respectively, displacement of the bound [[sup.3]H]adenosine from the NADH-SAH hydrolase under the assay conditions is unlikely. In addition, [N.sup.6] -methyladenosine, which often is used as internal standard for HPLC, shows a high affinity to NADH-SAH hydrolase ( [IC.sub.50] = 730 nmol/L).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
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; [S.sub.y|x] = 0.963).
[FIGURE 3 OMITTED]
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.
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 [micro]mol/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.
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 ([S.sub.y|x] = 1.12) for urine and 0.966 ([S.sub.y|x] = 0.92) for tissue, respectively.
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 hydroyse 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 [micro]g of protein per assay volume (300 [micro]L) is required to detect 10 nmol/L adenosine.
[FIGURE 4 OMITTED]
The binding assay with the NADH-SAH hydrolase and [[sup.3]H]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. 4B) 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.
[FIGURE 5 OMITTED]
Received October 5, 1999; accepted January 31, 2000.
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DORIS KLOOR,  * KOZO YAO,  URSULA DELABAR,  and HARTMUT OSSWALD 
 Department of Pharmacology, Faculty of Medicine, University of Tubingen, Wilhelmstrasse 56, D-72074 Tubingen, Germany.
 Department of Pharmacology, Kyowa Hakko Kogyo Co. Ltd, Mishima 411-8731, Japan.
* Author for correspondence. Fax 49-07071-294942; e-mail firstname.lastname@example.org.
Table 1. [IC.sub.50] values of 3 nmol/L [[sup.3]H]adenosine displacement using NAD1- and NADH-SAH hydrolase. [IC.sub.50], [micro] mol/L Competitor [NAD.sup.+]-SAHH (a) NADH-SAHH Adenosine 0.047 0.033 cAMP 0.104 70.0 Adenine 11.5 >100.0 2'-Deoxyadenosine 0.094 0.63 SAH 0.25 2.6 Homocysteine 5.0 5.0 AMP 10.0 >1000.0 ADP 10.0 >1000.0 ATP 10.0 >1000.0 Inosine 10.0 10.0 cGMP >1000.0 >1000.0 Diadenosine diphosphate 0.18 10.0 [N.sup.6]-Methyladenosine 0.09 0.73 (a) SAHH, SAH hydrolase. Table 2. Dilution linearity. Adenosine, nmol/L Dilution (a) Calculated Observed Recovery, % 0 311.0 1:03 103.7 99.6 96 1:10 31.1 28.7 92.3 1:30 10.4 9.1 87.5 1:100 3.1 3.4 109.7 (a) A urine sample containing 311.0 nmol/L adenosine was diluted in 20 mmol/L Tris-40 mmol/L HEPES, pH 7.4, to the concentration indicated. Table 3. Recovery of adenosine added to rat urine (samples 1 and 2), human urine (sample 3), and tissue (sample 4). Added Calculated Observed Recovery, % Sample 1 0 86.8 50 136.8 135.0 96.4 100 186.8 190.8 104.0 200 286.8 294.0 103.6 Sample 2 0 47.4 50 97.4 104.2 107.0 100 147.4 149.7 102.3 200 247.4 242.4 97.5 Average (CV) 101.8 (4.0%) Sample 3 0 3700 100 3800 3820 100.5 520 4220 4300 102.0 960 4660 4500 96.6 Average (CV) 99.7 (2.8%) Sample 4 0 20 700 100 20 800 20 650 99.2 500 21 200 21 350 100.7 1000 21 700 22 000 101.3 Average (CV) 100.4 (1.1%) Table 4. Intra- and interassay imprecision of the method. CV, % Sample adenosine, nmol/L Intraassay Interassay 32.1 3.6 7.4 47.4 1.2 3.0 97.0 2.3 5.9 4400 3.9 7.8 20 700 2.1 3.8
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|Title Annotation:||Automation and Analytical Techniques|
|Author:||Kloor, Doris; Yao, Kozo; Delabar, Ursula; Osswald, Hartmut|
|Date:||Apr 1, 2000|
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