Clinical Chemistry 46: 537-542, 2000;
(Clinical Chemistry. 2000;46:537-542.)
© 2000 American Association for Clinical Chemistry, Inc.
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
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Abstract
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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.
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Introduction
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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.
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Materials and Methods
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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.
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Results
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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 ( ) 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 ().
NADH-SAH hydrolase was incubated with 3 nmol/L
[3H]adenosine for various lengths of time.
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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.
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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.
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).
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).
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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.
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.
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Discussion
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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. 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.
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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.
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Adenosine and kidney function.
Physiol Rev,
July 1, 2006;
86(3):
901 - 940.
[Abstract]
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Abstract
Introduction
