Scand. 1. d i n . Lab. Invest., Vol. 36, 1976.
A Protein-Binding Assay for Direct Determination of Adenosine 3’,5’-Monophosphatein Amniotic Fluid, Cerebrospinal Fluid, Plasma, and Urine
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L. AKSNES & 0. SOVIK Dept. of Pediatrics, University of Bergen, Bergen, Norway
Aksnes, L. & Sovik, 0. A Protein-Binding Assay for Direct Determination of Adenosine 3’,5’-Monophosphate in Amniotic Fluid, Cerebrospinal Fluid, Plasma, and Urine. Scand. J. elin. Lab. Invest. 36, 289-298, 1976. An adenosine 3’,5’-monophosphate (cyclic AMP)-binding protein was isolated from bovine skeletal muscle. This preparation showed maximum binding capacity for cyclic AMP at the physiological pH of amniotic fluid, cerebrospinal fluid, and plasma and had a high association constant of 1.4 x lo9 l/mol. This preparation of binding protein, together with albumin and EDTA in the assay buffer, gave a sensitive and specific competitive protein-binding assay that permitted direct determination of cyclic AMP in the biological fluids mentioned above. Key-words: Competitive protein-binding assay; cyclic nucleotides; saturation assay L. Aksnes, Dept. of Pediatrics, University of Bergen, 5016 Haukeland sykehus, Bergen, Norway
Adenosine 3’,5’-monophosphate (cyclic AMP) is an important mediator of the effect of a variety of hormones and other biologically active agents. Cyclic AMP is found in nearly all types of mammalian cells and in extracellular fluids. Estimation of this cyclic nucleotide in extracellular I’luids seems to reflect a variety of intracellular processes, and the clinical value of such estimations is growing (5, 15, 24). Cyclic AMP has been measured by a variety of methods (14), but the most widely used are the competitive binding methods based on antibodies or naturally occurring binding proteins. Antibodies are made with a succinyl derivative of cyclic AMP bound to a carrier protein as immunogen (26). Naturally occurring binding proteins are easily prepared from adrenal tissue (8, 29) c r muscle (13). A problem in competitive binding assays is that of nonspecific interference, and in the case of the cyclic AMP assays compounds such as salts, metal ions, proteins, and nucleotides, all present in biological systems, have been shown to interfere. In the case of urine (cyclic AMP level, mol/l) the interference can be reduced or eliminated by 6 - Scand. J. clin. Lab. Invest.
diluting the samp!e. The levels of cyclic AMP in amniotic fluid, cerebrospinal fluid, and plasma are low (about mol/l) and do not permit extensive dilution. The problem of nonspecific interference has therefore been solved by different extraction methods (1, 10) and/or chromatographic isolation of cyclic AMP (6, 16, 22, 28), involving complex internal standardization methods. In the case of plasma, cyclic-AMP-free plasma has been added in preparing the calibrationcurve(3,9,17), but different plasma specimens are reported to give different nonspecific effects (3, 9), thus requiring calibration with a plasma blank for each sample. The purpose of the present work was to develop a simple and rapid assay for measurement of cyclic AMP in a large number of samples The method of choice was a direct measurement without timeconsuming extraction or separation procedures. These requirements were met by the isolation of a binding protein with high affinity for cyclic AMP at conditions that prevented precipitation of proteins and other components of the samples, and which gave a minimal nonspecific interference.
290
L. Aksnes & 0.Sovik
natant was adjusted to pH 4.8 by adding a 2 mol/l solution of acetic acid and was centrifuged at [3H]adenosine 3',5'-monophosphate ammonium 10,oOO g for 50 min. The supernatant was then salt ([3H]cyclic AMP) with a specific activity of adjusted to pH 6.8 by adding a 1 mol/l potassium38.4 Ci/mmol was obtained from New England phosphate buffer, pH 7.2, and 32.5 g (NHJ,S04 Nuclear (Boston, USA). Unlabeled adenosine was added per 100 ml. The precipitate formed 3',5'-monophosphate (cyclic AMP), guanosine after stirring for 7 h was collected by centrifuga3',5'-monophosphate (cyclic GMP), adenosine tion at 10,OOOg for 30 min, dissolved in 50 ml of 5'-monophosphate (AMP), adenosine triphos- 5 mmol/l potassium-phosphate buffer, pH 6.8, phate (ATP), and phosphodiesterase solution, with 2 mmol/l EDTA, and dialyzed extensively 0.15 Ujmg, were all obtained from Boehriinger against the same buffer. The dialyzed solution was Mannheim GmbH (Germany). Diethylamino- centrifuged at 12,000 g for 30 rnin and applied to ethyl-cellulose, DE-52, (DEAE-cellulose) was a 2 . 6 ~ 3 0cm column of DEAE-cellulose preobtained from Whatman Biochemicals Ltd. viously equilibrated against the dialyzing buffer. (Maidstone, England). AG 1-X8 resin, 200400 The column was washed with 500 ml of the same mesh (formate form), was obtained from Bio-Rad buffer, and the binding protein was eluted by a linear concentration gradient of potassiumLaboratories (Richmond, Cali., USA). Activated charcoal SP-1 was obtained from phosphate, pH 6.8, with 2 mmol/l EDTA, from 5 Serva Feinbiochemica GmbH & Co. (Heidelberg, to 500 mmol/l in 2 1 buffer. The eluting rate was Germany), and bovine serum albumin fraction V 45 ml/h, and 15-ml fractions were collected, read (BSA) from Sigma Chemical Co. (Saint Louis, in a spectrophotometer at 280 nm, and tested for USA). Polyethylenetest tubes, 15 x 100 mm, were cyclic AMP binding activity. Fractions in the first obtained from A/S Nunc (Roskilde, Denmark) peak of binding activity were pooled and dialyzed and Triton X-100, toluene, 2.5-diphenyloxazol against a 5 mmol/l potassium-phosphate buffer, (PPO), and dimethyl-p-bis (2-(5-diphenyl-oxa- pH 7.4, with 2 mmol/l EDTA. Portions were zoly1)-benzene) (dimethyl-POPOP) from Koch- stored at -20 "C. Light Laboratories Ltd. (Colnbrook, England). The binding reaction was performed by mixing 50 p1 [3H]cyclicAMP and 100 pl binding protein METHODS solution, both diluted in the assay buffer to the [3H]cyclic AMP was diiuted with distilled water desired concentration. Assay buffer was added to to a concentration of 50 nmol/l and storcd at a total volume of 300 or 350 pl. To prevent denaturation of the binding protein, the components -20 "C in small portions. Cyclic AMP, cyclic GMP, AMP, and ATP of the reaction mixture were gently mixed by hand were stored as stock solutions in distilled water before incubation. After 2 h, 100 pl of a charcoal at -20 "C. suspension was added. The charcoal suspension Binding protein was isolated from bovine contained 50 mg charcoal per ml assay buffer with skeletal muscle (beef tenderloin) mainly as de- 2% (wt/vol) BSA. The tubes were carefully shaken scribedby Miyamoto et al. (20) and Gilman (13). by hand and centrifuged for 5 min at 7,000 g. All Muscle tissue was obtained from a local slaughter- steps were performed at 2 "C or in an ice bath. house as soon as possibleafter slaughter,transpor- Two hundred and fifty pl of the supernatantswere ted on ice, and frozen at -20 "C before use. All placed in scintillationvials, 1 ml of distilled water isolation steps were performed at 4 "Cor in an was added, and the vials were filled up with 10 ml ice bath. of a scintillation mixture containing toluene and Two hundred and fifty g of muscle was cut into Triton X-100 (2:1, v/v), 5 % PPO, and 0.05% small pieces and homogenized with 750 ml of 50 dimethyl-POPOP.The vials were shaken until the mmol/l potassium-phosphate buffer, pH 7.0, with solution was clear. Counting was performed for 4 mmol/l EDTA in a Servall Omnimixerfor 3 min. 5 rnin in a Packard-Tri-Carb liquid scintillation The homogenate was filtered through glass-wool counter. and centrifuged at 10,OOOg for 40 min. The superStandard cyclic AMP assay was performed with
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MATERIAL
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Assayfor Cyclic AMP
a buffer containing 50 mmol/l potassium-phosphate and 2 mmol/l EDTA, pH 7.4, in the following referred to as standard assay buffer. The components were added in the following order: Fifty pl of a solution containing 0.5 pmol [3HH]cyclicAMP in standard assay buffer. Fifty p1 standard or unknown. Hundred pl binding protein solution in standard assay buffer with 1yo(wt/vol) BSA, and Standard assay buffer to a total volume of 350 p.1. In the case of calibration for plasma, cerebrospinal fluid, and amniotic fluid determinations, 50 $1 cyclic-AMP-free material was added before the addition of binding protein. The incubation, separation of free and bound [3H]cyclic AMP, and radioactive counting were performed as described under binding reaction. The counts per minute (c.p.m.) obtained in the presence of unlabeled cyclic AMP (Cx) and c.p.m. in the absence of unlabeled cyclic AMP (C,) were used for the construction of calibration curves by plotting the ratio Cx/Coagainst log concentration in nmol/l of cyclic AMP in the added standard solution. Blood, Cerebrospinal fluid, and amniotic fluid samples were collected by venipuncture, lumbar puncture, and amniocentesis, respectively, and EDTA was added immediately to give a final concentration of 5 mmol/l. The tubes were immediately mixed, centrifuged at 2 "C, and kept at -20 "Cuntil analyzed. Urine samples were frozen at -20 "C as soon as possible after collection. After thawing, before the analysis, the samples were centrifuged if there was any sign of turbidity. Standard assay buffer was added to the desired dilutions. Cyclic-AMP-free plasma, cerebrospinal fluid, and amniotic fluid were prepared from samples without added EDTA (blood samples collected in heparinized tubes). The samples were incubated with 0.03 U phosphodiesterase per ml for 15 h at 32 "C. The phosphodiesterase solution (0.1 5 U/mg protein) had previously been dialyzed against a 50 mmol/l potassium-phosphate buffer, pH 7.4, with 50 mmol/l magnesium sulfate. After incubation, EDTA was added to a final concentration of 5 mmol/l, and the samples were stored at -20 "C. Samples were also pooled for use in the assay.
291
Protein was determined by the method of Lowry et al. (19) and creatinine by the method of Bosnes 8c Taussky (7). Trichloroacetic acid (TCA) extraction was performed by mixing 500 pl of sample and 100 pl 300/, (wtlvol) TCA. After centrifugation, 400 $1 of the supernatant was acidified by adding 45 p1 of 1 mol/l HCl and extracted seven times with three volumes of water-saturated ether. The extract was dried at room temperature for 12 h under airstream and dissolved in standard assay buffer. Ion-exchange column purification of TCAextracted samples and diluted urine (1 :500) was performed on 0.4x3.5 cm AG 1-X8 formate columns, as described by Murad et al. (22). To correct for recovery, 0.01 pmol [3H]cyclic AMP was added to the samples before extraction and purification. The [3H]cyclic AMP used for recovery experiments was purified on an AG 1-X8 formate column before use to remove tritiated water formed during storage.
RESULTS AND DISCUSSlON Preparation of bindinfi protein
As shown in Fig. 1, three distinct peaks of binding activity were found in the eluate from the DEAEcellulose column, tested with both 50 mmol/l sodium acetate, pH 4.0, and 50 mmol/l potassiumphosphate, pH 7.4, as assay buffers. The fractions in peak I were pooled as indicated in Fig. 1, and this material was used in the binding studies mentioned below, and also in the assay system for cyclic AMP. The amount of binding protein obtained was about 80 mg. In the cyclic AMP assays previously reported, the first peak of binding activity from the DEAE-cellulose, eluted by the method of Gilman (13), was used without specification of the number of activity peaks found (11,27). In the work of Gilman (13) the first peak probably contains a mixture of peaks I and I1 in the present work (Fig. 1). The improvement in separation of peaks 1 and I1 was obtained by lowering the pH from 7.0 to 6.8. p H dependence of the cyclic AMP binding As shown in Fig. 2, there was a marked increase in apparent cyclic AMP binding when the pH was increased from 4.0 to 5.0. When the p H was
L. Aksnes & 0.Sovik
292 I\
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40W
Frxtcon no
Fig. 1. DEAE-cellulose chromatography of binding protein. Determination of binding activity in the fractions (15 ml) was carried out as described in the text with 0.5 pmol LH-labeled 3’,5’-monophosphate (cyclic AMP) and 10 p1 of the fractions. A: (O---O)=relative binding activity with 50 mmol/l sodium acetate, pH 4.0, as assay buffer; (---)== potassium phosphate gradient; +--/)=pooled fractions used in the assay. B: (.---.)=relative binding activity with 50 mmol/l potassium phosphate, pH 7.4, as assay buffer; (x-x)=optical density at 280 nm. further increased to 6.2, there was a drop in binding capacity, whereas a clearly improved binding was observed by raising the pH from b.2 to 7.7. Using a potassium-phosphate buffer, there was only a small variation in binding between pH 7 and 8. Somewhat higher binding activity was observed with potassium-phosphate than with Tris-HC1 buffer. For comparison the protein kinase preparation of Gilman (13) and an adrenal protein used by Tsang et al. (ZS), both using acetate buffer, gave maximal binding at pH 4.0 and 5.0, respectively. Nonspecific interference The interference in the standard cyclic AMP assay by CaCl,, MgClZ, NaCl, BSA, plasma, cerebrospinal fluid, amniotic fluid, and diluted
Fig. 2. Effect of pH on adenosine 3’,5’-monophosphate (cyclic AMP) binding activity. Binding was carried out as described in the text using 5 pg binding protein, 0.5 pmol [sH]cyclicAMP, and 50 mmol/l assay buffer, to a total volume of 300 pl. Assay buffers used were (0--O)=sodium acetate acetic acid, (.---.)= potassium phosphate, ( X ---x)=Tris-HCl. urine at different concentrations of unlabeled cyclic AMP is shown in Table I. Albumin up to lo%, NaCl up to 0.5 mol/l, and urine and the divalent cations Ca2+and Mg2+at concentrations up to 5 mmol/l did not interfere at any concentration of unlabeled cyclic AMP. At 7.5 mmol/l and above Ca2f and Mg2+caused a decrease in bound labeled cyclic AMP. The degree of nonspecific interference was dependent on the concentration of unlabeled cyclic AMP in the reaction mixture. This was also observed by Albano et al. (1). Plasma, cerebrospinal fluid, and amniotic fluid caused a small increase in bound labeled cyclic AMP at all concentrations of unlabeled cyclic AMP. Different samples of plasma, cerebrospinal fluid, and amniotic fluid did not show any variation in interference. The same increase was also observed after diluting these samples 1:l with assay buffer. The effect of cyclic GMP, ATP, and AMP on the binding of cyclic AMP is shown in Fig. 3. Cyclic G M P showed the most marked interference, but a concentration about 1 0 0 times that of cyclic AMP was necessary to give the same decrease in [3H]cyclicAMP binding. ATP had no effect at concentrations below 5 >c lop4mol/l, and
Assay for Cyclic AMP
293
Table I. Binding of 3H-labeled adenosine 3‘,5’-monophosphate (cyclic AMP) at different concentrations of unlabeled cyclic AMP made up in different media. Binding studies were performed with 0.5 pmol [3H]cyclic AMP and 50 ~1of the indicated media as described under standard cyclic AMP assay in the text Medium (concentration of cyclic AMP in medium, nmol/l)
[3H]cyclicAMP bound, pmol, rneanfS.D. (n=4) 6
0
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~~
Assay buffer CaCl,, 5 mmol/l CaCl,, 7.5 mmol/l MgClz, 5 mmolil MgCl,, 7.5 mmol/l NaCl, 0.2 mol/l NaCI, 0.5 mol/l BSA, 10% Mixture containing CaCl,, 5 mmol/l; MgCl,, 5 mmol/l; NaCl, 0.2 mol/l; and BSA, lo?; Plasma Cerebrospinal fluid Amniotic fluid Urine (1:lOO)
O3
i
25 ~~
0.272*0.007 0.270+0.008 0.248*0.010 0.268*0.008 0.252+0.008 0.279+0.005 0.273f0.008 0.270f0.004
0.229&0.006 0.231 50.007 0.211&0.007 0.227*0.007 0.209+0.007 0.235+0.007 0.230f0.006 0.227f0.005
0.127f0.003 0.125fO.OO4 0.122&0.006 0.12450.005 0.123*O.OO4 0.12510.003 0.130&0.005 0.130+0.003
0.270&0.006 0.285*0.006 0.282&0.002 0.28410.006 0.268&0.008
0.227&0.005 0.247&0.005 0.243&0.004 0.245f0.005 0.232+0.006
0.130~0.004 0.140&0.003 0.139&0.004 0.137f0.002 0.125+0.003
AMP had no effect below 10V mol/l. In conclusion Ca2’, Mg2+,NaCl, and protein (BSA) had no effect on the cyclic AMP binding within the concentration limits found in extracellular fluids. The specificity of the binding reaction is high, and the cross-reactivity of the binding reaction by cyclic G M P and ATP is no problem in view of the relative levels of cyclic AMP and these nucleotides in biological materials.
AMP
Standard assay conditions
O
,o‘B
U
’ K)-7
a
K)-5
,o.L
nucleotide concentration
-
lo-3
mol
,o-2
/I
Fig. 3. Binding of 3H-labeled 3’,5’-monophosphate (cyclic AMP) in the presence of cyclic AMP, guanoshe 3’,5’-monophosphate (cyclic GMP), adenosine 5’monophosphate (AMP), and adenosine triphosphate (ATP). Binding was carried out as described under standard cyclic AMP assay in the text with 50 p1 of the nucleotide solutions.
Fifty mmol/l potassium phosphate, pH 7.4, was chosen as the standard assay buffer because its p H is very close to that of plasma, cerebrospinal fluid, and amniotic fluid and to the p H where the binding of cyclic AMP isnear maximum. EDTA was added to inhibit the Mg2+-dependent phosphodiesterase present in the samples (3) and to reduce the effect of metal ions on the binding reaction ar,d separation of bound and frec cyclic AMP by charcoal (1, 13). Gilman (13) reported that addition of a protein kinase inhibitor isolated from bovine skeletal muscle increased both the affinity with cyclic AMP and the number of available binding sites on his protein kinase preparation. The same effect was later observed with other protein preparations, among them BSA (21). The effect
294
L. Aksnes & 0.Sovik
t
V
4 Q
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f
o.61 05
5
p9
bmdq Wtan
Fig. 4. Double. reciprocal plots of adenosine 3’,5’monophosphate (cyclic AMP) binding. Binding was carried out as described in the text in a total volume of 300 d,using 5 pg binding protein and 50 mmol/l potassium phosphate, pH 7.4, with 2 mmol/l EDTA as assay buffer. (-)=with 1 mg bovine serum albumin (BSA) in the reaction mixture, (O-o)= without BSA.
Fig. 5. Effect of dilution of binding protein on the binding of adenosine 3’,5’-monophosphate (cyclic AMP). Binding was carried out as described under standard cyclic AMP assay in the text with 0.5 pmol [aHlcyclic AMP.
of BSA on the binding properties of the binding protein was investigated also in the present work and was found to activate the binding protein and neutralize the degree of nonspecific effects an the binding due to differences in composition of biological fluids. The optimal effect was obtained by using 1 mg BSA in the reaction mixture (350 ~ 1 ) . The binding, in presence and absence of BSA, was evaluated by double reciprocal plots of bound against free cyclic AMP, as shown in Fig. 4.The association constant was increased from 1.4 to 1.8 x los l/mol, and the number of available sites was also increased. One pg binding protein was found to bind 0.13 and 0.08 pmol cyclic AMP in presence and absence of BSA, respectively. The binding data obtained are the sum of two reactions, the binding of cyclic AMP to the binding protein and the separation of free and bound cyclic AMP. The different methods used for separation of free and bound cyclic AMP are all shown to be more or less influenced by environmental factors. The membrane filtration method is influenced by EDTA, metal ions, and proteins (4, 28, 31). The ion-exchange method is influenced by salts and plasma extracts and gives incomplete uptake of cyclic AMP(28). The charcoal method is influenced
by metal ions (1) and plasma extracts (28), but none of these factors were found to influence the separation in the present study. The amount and grade of charcoal were found to be important factors for satisfactory separation (9). With the amount and grade used in the present study, 99.5% of the free cyclic AMP was absorbed. Maximum binding of [3H]cyclic AMP obtained was 88%. The time elapsed from addition of charcoal to centrifugation was found to be important, probably due to dissociation of the bound cyclic AMP. A nearly linear decrease (about 5%) in bound cyclic AMP was found within the first 10 min. The time factor for removal of supernatant was less important, since no difference was found in specimens taken within the first 10 min after the centrifuge was stopped. The charcoal method is inexpensive and convenient to use for a large number of samples. A saturating concentration of cyclic AMP was chosen because this gave the most precise and sensitive assay with a minimum of interference from other substances. The amount of binding protein chosen was that required to give 50% binding of the 0.5 pmol [“HJcyclicAMP added,
Assay for Cyclic AMP
295
the whole concentration range of cyclic AMP measured. Calibration with a cyclic-AMP-free pool of the same type of fluid that was to be tested was preferred, since this gave an improved reproducibility of the results.
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Plasma sumplts
01
02 0 3
04
05
06
07
08
09
10
W C O
Fig. 6. Typical calibration curve for the assay of adenosine 3’,5’-monophosphate (cyclic AMP). The abscissa shows the ratio of c.p.m. in the presence of unlabeled cyclic AMP (C,) and c.p.m. in the presence of unlabeled cyclic AMP (CO).The ordinate shows the concentration in nmol/l added standard cyclic AMP solution (50 pl) on a logarithmic scale. calculated from a plot shown in Fig. 5. Fig. 6 shows a typical calibration curve. The semilogarithmic plot gave a nearly linear curve between the concentrations 6 and 50 nmol/l. The small nonspecific interference by plasma, cerebrospinal fluid, and amniotic fluid can be corrected for by simple subtraction because the interference was found to be nearly constant for different samples of the same type of fluid over
For the direct determination of cyclic AMP in plasma samples calibration was carried out by addition of 50 pl of a cyclic-AMP-free plasma pool. Addition of cyclic-AMP-free plasma had no effect on the Cx/Co ratio, and between-assay precision was within the level for determinations in assay buffer only (Table 11). The coefficient of variation was less than 7% within the range 6-50 nmol/l for duplicate determinations with six different cyclic-AMP-free plasma samples used fcr calibration. Table I11 shows the validity of the assay. The sample can be diluted 1 : 1 without changing the degree of nonspecific interference, which indicates that the assay conditions neutralize variations in compositions of the various samples. The assay also seems to be free of the reported nonspecific interference caused by the TCA extraction method (1). The immediate addition of EDTA to the samples is important because of the high phosphodiesterase activity, even in frozen samples (3). After addition of EDTA to a final concentration of 5 mmol/l, no decrease in cyclic AMP was observed after 15 h of incubation at 32 “C. Plasma cyclic AMP measured in 10 normal
Table 11. Between-assay reproducibility and precision in the standard adenosine 3’,5’-monophosphate (cyclic AMP) assay for the ratio between c.p.rn. in the presence of unlabeled cyclic AMP (Cx) and c.p.m. in the absence of unlabeled cyclic AMP (C,), and determination of cyclic AMP in nrnol/l. Cyclic AMP standards were assayed on 12 separate days Cyclic AMP, nmol/l
CXICO Cyclic AMP, nmol/l (50 pl) 3 6 12 25 50
100 200
Meanf S.D. (n=12) 0.891&0.020 0.76510.017 0.604&0.018 0.402*0.020 0.225+0.021 0.131 10.015 0.079+0.014
Coefficient of variation (yo) 2.2 2.2
3.0 4.9 9.3 11.5 17.7
S.D. (n-12)
-- 0.3
Coefficient of variation (96)
-- 0.4
9.7 6.8
- 0.7 - 1.8 2.5 Zll.0
-
7.2 5.0 11.0
130.0
15.0
5.6
296
L. Aksnes & 0.Sovik
Table 111. Direct measurements of adenosine 3',5'-monophosphate (cyclic AMP) in plasma, cerebrospinal fluid (CSF), and amniotic fluid (AF) and measurements of trichloroaceticacid (TCA) extracts and ionexchangepurified extracts of the same fluids. Assay was carried out as described under standard cyclic AMP assay in the text, calibrated with standard assay buffer (+)or cyclic-AMP-free pool of the assayed fluid (++)
pmol cyclic AMP added per ml sample
Preparation
Plasma
CSF
AF
Cyclic-AMP-free Cyclic-AMP-free, diluted 1:1 Cyclic-AMP-free; TCAextracted + Cyclic-AMP-free, ionexchange
10 20 20
19.6f0.9 10.2f0.5 20.1 f1.5 18.8f2.1
20.751.0 9.6f0.7 19.1k2.3 20.5f2.4
19.811.2 10.510.7 19.351.7 19.9f1.9
Collected in EDTA Collected in EDTA, diluted 1:1 -t Collected in EDTA, TCAextracted Collected in EDTA, ionexchange
0 0 0 0
20.3h0.9 10.4f0.5 19.8f1.6 21.0f1.7
22.1f1.4 11.6f0.6 21.4f 1.7 21.3f2.0
30.71 1.4 14.9f0.7 29.1 52.1 31.012.6
20
+ +
++
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Measured cyclic AMP in sample in nmol/l, meanfS.D. (n=4)
+
+ +
+
+
+
young adults (6 females and 4 males) gave a mean of 17.7 nmol/l f 2.5 (S.E.M.)(range, 12.5 to 22.0).
good agreement with reported values in this patient group (18).
Amniotic fluid
Calibration for direct determination in cerebrospinal fluid was performed with 50 pl of a cyclicAMP-free cerebrospinal fluid pool. The betweenassay coefficient of variation was less than 6.5% within the range 6-50 nmol/l for duplicate determinations with six different cerebrospinal fluids used for calibration. The data in Table I11 show good agreement of determinations in undiluted samples, samples diluted 1: 1, samples extracted by TCA, and samples purified on ion-exchange columns. Cerebrospinal fluid samples from 10 patients aged 6-13 years with acute leukemia, taken during routine control of the disease, gave a mean of 21.2 nmo1,'l & 2.0 (S.E.M.) (range, 17.5-24.6), which is in good agreement with values reported elsewhere (23, 25).
For the direct determination of cyclic AMP in amniotic fluid calibration was carried out with the addition of 50 pl of a cyclic-AMP-free amniotic fluid pool. The between-assay coefficient of variation was less than 7.5% within the range of 6 to 50 nmol/l for duplicate determinations with seven different cyclic-AMP-free amniotic fluids used for calibration. The data in Table IIL show good agreement between determinations in undiluted samples, samples diluted 1:1, samples extracted by TCA, and samples purified by ion-exchange chromatography. Although the composition of amniotic fluid changes during pregnancy (l'), samples from different stages of gestation (22-40 weeks) caused no variation in nonspecific interference. The half-life of cyclic AMP in amniotic fluid incubated at 32 "C was 7-10 h, indicating a rapid degradation of the nucleotide. Degradation was also observed in frozen samples (-20 "C). Addition of EDTA prevented the degradation, indicating the presence of a Mg2+-dependent phosphodiesterase. This enzyme activity has not previously been reported in amniotic fluid. A value of 45.6 nmol/lf7.5 (S.E.M.) was found in amniotic fluids from pregnancies complicated by hypertension, which is in
Cerebrospinal fluid
Urine
Diluted urine samples did not bring any problem of nonspecific interference and were consequently assayed directly without addition of cyclic-AMP-free urine for calibration. The urine samples were usually diluted 1 :500. Urine from 20 normal children aged 2-10 years gave a mean value of 5.9 h 1.2 (S.E.M.) nmol cyclic AMP per mg creatinine (range, 2.5- l0.5),
Assay for Cyclic AMP which is in good agreement with values reported by August et al. (2).
ACKNOWLEDGEMENTS This work u a s aided by grants from the Nordic Insulin Fund and from the Naiisen Foundation.
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REFERENCES 1. Albano, J. D. M., Barnes, G. D., Maudsley, D. V., Brown, B. L. & Ekins, R. P. Factors affecting the saturation assay of cyclic AMP in biological systems. Analyr. Biochem. 60, 130, 1974. 2. August, G. P., Hung, W. & Rivard, B. The urinary excretion of adenosine 3’,5’-monophosphate in children and adolescents. J. clin. Endocr. 37, 476, 1973. 3. Barling, P. M., Albano, J. D. M., Tomlinson, S., Brown, B. L. & O’Riordan, J. L. H. A saturation assay for adenosine 3’:5’cyclic monophosphate in plasma and its use in studies of the action of bovine parathyroid hormone. Biochem. SOC. Trans. 2, 453, 1974. 4. Binoux, M. A. & Odell, W. D. Use of dextran coated charcoal to separate antibody-bound from free hormone. J . clin. Endocr. 36, 303, 1973. 5. Bitensky, M. W., Keirns, J. J. & Freeman, J. Cyclic adenosine monophosphate and clinical medicine. I. Amer. J . med. Sci. 266, 320, 1973. 6. Booker, G., Thomas, L. J. & Appleman, M. M. The assay of adenosine 3’,5’-cyclic monophosphate and guanosine 3’,5’-cyclic monophosphate in biological materials by enzymatic radioisotopic displacement. Biochemistry (Wash.) 7, 4177, 1968. 7. Bosnes, R. W. & Taussky, H. H. On the colorimetric determination of creatinine by the Jaffe reaction. J . biol. Chem. 158, 581, 1945. 8 . Brown, B. L., Albano, J. D. M., Ekins, R. P. & Sgherzi, A. M. A simple and sensitive saturation assay method for the measurement of adenosine 3‘5’-cyclic monophosphate. Biochem. J . 121, 561, 1971. 9. Brown, B. L., Ekins, R. P. & Albano, J. D. M. Saturation assay for cyclic AMP using endogenous binding protein. pp. 25-40 in Greengard, P. & Robison, G. A. (eds.) Advances in Cyclic Nucleoride Research, Vol. 2. Raven Press, New York, 1972. 10. Chan, P. S., Black, C. T. & Williams, B. J. Separation of cyclic 3’,5’-AMP from ATP, ADP and 5’-AMP by precipitation with inorganic compounds. Analyt. Biochem. 55, 16, 1973. 11. Cooper, R. H., Ashcroft, S. J. H. & Randle, P. J. Concentration of adenosine 3’:5’-cyclic monophosphate in mouse pancreatic islets measured by a protein-binding radioassay. Biochem. J . 134, 599, 1973.
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12. Fairweather, D. V. I. & Eskes, T. K. A. B. (eds.) Amniotic Fluid, Research and Clinical Application. Excerpta Media, Amsterdam, 1973. 13. Gilman, A. G. A protein binding assay for sd:nosine 3’5’-cyclic monophosphate. Proc. nut. Acad. Sci. (Wash.)67, 305, 1970. 14. Greengard, P. & Robison, G. A. (eds.) Advances in Cyclic Nucleoride Research, Vol. 2. Raven Press, New York, 1972. 15. Keirns, J. J., Freeman, J. & Bitensky, M. W. Cyclic adenosine monophosphate and clinical medicine. 11. Amer. J. med. Sci. 268, 62, 1974. 16. Krishna, G. & Birnbaumer, L. On the assay of adenyl cyclase. Analyt. Biochem. 35, 393, 1970. 17. Latner, A. L. & Prudhoe, K. A simplified competitive proteinbinding assay for adenosine 3’3’monophosphate in plasma. Clin. chim. Acra 48, 353, 1973. 18. Ling, W. Y., Marsh, J. M., Spellacy, W. N., Tresher, A. J. & Lemaire, W. J. Adenosine 3’3’monophosphate in amniotic fluid from pregnancy complicated by hypertension. J . clin. Endocr. 39, 479, 1974. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with Folin phenol reagent. J. biol. Chem 193,256, 1951. 20. Miyamoto, E., Kuo, J. F. & Greengard, P. Cyclic nucleotide-dependent protein kinases. 111. Purification and properties of adenosine 3’,5’-monophosphate-dependent protein kinases from bovine brain. J . biol. Chem. 244, 6395, 1969. 21. Murad, F. & Gilman, A. G. Adenosine 3’3’monophosphate and guanosine 3‘,5‘-monophosphate : a simultanous protein binding assay. Biochim. biophys. Acta (Amst.) 252, 397, 1971. 22. Murad, F., Manganiello, V. & Vaughan, M. A simple, sensitive protein binding assay for guanosine 3’,5‘-monophosphate. Proc. nut. Acad. Sci. (Wash.) 68, 736, 1971. 23. Myllyla, V. V., Vapaatalo, H., Hokkanen, E. & Heikkinen, E. R. Cerebrospinal fluid concentration of cyclic adenosine 3’,5’-monophosphate and pneumoencephalography. Europ. Neurol. 12, 28, 1974. 24. Oye, I. Cyclic AMP and its relation to clinical chemistry. Scand. J. clin. Lab. Invest. 32, 189, 1973. 25. Robison, G. A., Coppen, A. J., Whybrow, P. C. & Prange, A. J. Cyclic A.M.P. in affective disorders. Lancer 2, 1028, 1970. 26. Steiner, A. L., Kipnis, D. M., Utiger, R. & Parker, C. Radioimmunoassay for the measurement of adenosine 3’,5’-cyclic phosphate. Proc. nut. Acad. Sci. (Wash.)64, 367, 1969. 27. Tovey, K. C., Oldham, K. G. & Whelan, J. A. M. A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique. Clin. chim. Acta 56, 221, 1974.
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28. Tsang, C. P. W., Lehotay, D. C. & Murphy, B. E. P. Competitive binding assay for adenosine 3',5'monophosphate employing a bovine adrenal protein. Application to urine, plasma and tissue. J. clin. Endocr. 35, 809,1972. 29. Walton, G. M. & Garren, L. D. An assay for adenosine 3',5'-cyclic monophosphate based on the association of the nucleotide with a partially purified binding protein. Bioehertistry (Wash.) 9, 4223, 1970.
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Received 25 September 1975 Accepted 22 Dwmber 1975
30. White, A. A. & Zenser, T. V. Separation of cyclic 3',5' nucleoside monophosphates from the other nucleotides on alumina oxide columns. Application to the assay of adenyl cyclase and guanyl cyclase. Analyf.Biochem. 41, 372, 1971. 31.Yarus, M. & Berg, P. On the properties and utility of a filter assay in the study of isoleucyltRNA syntethase. Analyt. Biochem. 35,450, 1970.
A Protein-Binding Assay for Direct Determination of Adenosine 3’,5’-Monophosphatein Amniotic Fluid, Cerebrospinal Fluid, Plasma, and Urine
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L. AKSNES & 0. SOVIK Dept. of Pediatrics, University of Bergen, Bergen, Norway
Aksnes, L. & Sovik, 0. A Protein-Binding Assay for Direct Determination of Adenosine 3’,5’-Monophosphate in Amniotic Fluid, Cerebrospinal Fluid, Plasma, and Urine. Scand. J. elin. Lab. Invest. 36, 289-298, 1976. An adenosine 3’,5’-monophosphate (cyclic AMP)-binding protein was isolated from bovine skeletal muscle. This preparation showed maximum binding capacity for cyclic AMP at the physiological pH of amniotic fluid, cerebrospinal fluid, and plasma and had a high association constant of 1.4 x lo9 l/mol. This preparation of binding protein, together with albumin and EDTA in the assay buffer, gave a sensitive and specific competitive protein-binding assay that permitted direct determination of cyclic AMP in the biological fluids mentioned above. Key-words: Competitive protein-binding assay; cyclic nucleotides; saturation assay L. Aksnes, Dept. of Pediatrics, University of Bergen, 5016 Haukeland sykehus, Bergen, Norway
Adenosine 3’,5’-monophosphate (cyclic AMP) is an important mediator of the effect of a variety of hormones and other biologically active agents. Cyclic AMP is found in nearly all types of mammalian cells and in extracellular fluids. Estimation of this cyclic nucleotide in extracellular I’luids seems to reflect a variety of intracellular processes, and the clinical value of such estimations is growing (5, 15, 24). Cyclic AMP has been measured by a variety of methods (14), but the most widely used are the competitive binding methods based on antibodies or naturally occurring binding proteins. Antibodies are made with a succinyl derivative of cyclic AMP bound to a carrier protein as immunogen (26). Naturally occurring binding proteins are easily prepared from adrenal tissue (8, 29) c r muscle (13). A problem in competitive binding assays is that of nonspecific interference, and in the case of the cyclic AMP assays compounds such as salts, metal ions, proteins, and nucleotides, all present in biological systems, have been shown to interfere. In the case of urine (cyclic AMP level, mol/l) the interference can be reduced or eliminated by 6 - Scand. J. clin. Lab. Invest.
diluting the samp!e. The levels of cyclic AMP in amniotic fluid, cerebrospinal fluid, and plasma are low (about mol/l) and do not permit extensive dilution. The problem of nonspecific interference has therefore been solved by different extraction methods (1, 10) and/or chromatographic isolation of cyclic AMP (6, 16, 22, 28), involving complex internal standardization methods. In the case of plasma, cyclic-AMP-free plasma has been added in preparing the calibrationcurve(3,9,17), but different plasma specimens are reported to give different nonspecific effects (3, 9), thus requiring calibration with a plasma blank for each sample. The purpose of the present work was to develop a simple and rapid assay for measurement of cyclic AMP in a large number of samples The method of choice was a direct measurement without timeconsuming extraction or separation procedures. These requirements were met by the isolation of a binding protein with high affinity for cyclic AMP at conditions that prevented precipitation of proteins and other components of the samples, and which gave a minimal nonspecific interference.
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natant was adjusted to pH 4.8 by adding a 2 mol/l solution of acetic acid and was centrifuged at [3H]adenosine 3',5'-monophosphate ammonium 10,oOO g for 50 min. The supernatant was then salt ([3H]cyclic AMP) with a specific activity of adjusted to pH 6.8 by adding a 1 mol/l potassium38.4 Ci/mmol was obtained from New England phosphate buffer, pH 7.2, and 32.5 g (NHJ,S04 Nuclear (Boston, USA). Unlabeled adenosine was added per 100 ml. The precipitate formed 3',5'-monophosphate (cyclic AMP), guanosine after stirring for 7 h was collected by centrifuga3',5'-monophosphate (cyclic GMP), adenosine tion at 10,OOOg for 30 min, dissolved in 50 ml of 5'-monophosphate (AMP), adenosine triphos- 5 mmol/l potassium-phosphate buffer, pH 6.8, phate (ATP), and phosphodiesterase solution, with 2 mmol/l EDTA, and dialyzed extensively 0.15 Ujmg, were all obtained from Boehriinger against the same buffer. The dialyzed solution was Mannheim GmbH (Germany). Diethylamino- centrifuged at 12,000 g for 30 rnin and applied to ethyl-cellulose, DE-52, (DEAE-cellulose) was a 2 . 6 ~ 3 0cm column of DEAE-cellulose preobtained from Whatman Biochemicals Ltd. viously equilibrated against the dialyzing buffer. (Maidstone, England). AG 1-X8 resin, 200400 The column was washed with 500 ml of the same mesh (formate form), was obtained from Bio-Rad buffer, and the binding protein was eluted by a linear concentration gradient of potassiumLaboratories (Richmond, Cali., USA). Activated charcoal SP-1 was obtained from phosphate, pH 6.8, with 2 mmol/l EDTA, from 5 Serva Feinbiochemica GmbH & Co. (Heidelberg, to 500 mmol/l in 2 1 buffer. The eluting rate was Germany), and bovine serum albumin fraction V 45 ml/h, and 15-ml fractions were collected, read (BSA) from Sigma Chemical Co. (Saint Louis, in a spectrophotometer at 280 nm, and tested for USA). Polyethylenetest tubes, 15 x 100 mm, were cyclic AMP binding activity. Fractions in the first obtained from A/S Nunc (Roskilde, Denmark) peak of binding activity were pooled and dialyzed and Triton X-100, toluene, 2.5-diphenyloxazol against a 5 mmol/l potassium-phosphate buffer, (PPO), and dimethyl-p-bis (2-(5-diphenyl-oxa- pH 7.4, with 2 mmol/l EDTA. Portions were zoly1)-benzene) (dimethyl-POPOP) from Koch- stored at -20 "C. Light Laboratories Ltd. (Colnbrook, England). The binding reaction was performed by mixing 50 p1 [3H]cyclicAMP and 100 pl binding protein METHODS solution, both diluted in the assay buffer to the [3H]cyclic AMP was diiuted with distilled water desired concentration. Assay buffer was added to to a concentration of 50 nmol/l and storcd at a total volume of 300 or 350 pl. To prevent denaturation of the binding protein, the components -20 "C in small portions. Cyclic AMP, cyclic GMP, AMP, and ATP of the reaction mixture were gently mixed by hand were stored as stock solutions in distilled water before incubation. After 2 h, 100 pl of a charcoal at -20 "C. suspension was added. The charcoal suspension Binding protein was isolated from bovine contained 50 mg charcoal per ml assay buffer with skeletal muscle (beef tenderloin) mainly as de- 2% (wt/vol) BSA. The tubes were carefully shaken scribedby Miyamoto et al. (20) and Gilman (13). by hand and centrifuged for 5 min at 7,000 g. All Muscle tissue was obtained from a local slaughter- steps were performed at 2 "C or in an ice bath. house as soon as possibleafter slaughter,transpor- Two hundred and fifty pl of the supernatantswere ted on ice, and frozen at -20 "C before use. All placed in scintillationvials, 1 ml of distilled water isolation steps were performed at 4 "Cor in an was added, and the vials were filled up with 10 ml ice bath. of a scintillation mixture containing toluene and Two hundred and fifty g of muscle was cut into Triton X-100 (2:1, v/v), 5 % PPO, and 0.05% small pieces and homogenized with 750 ml of 50 dimethyl-POPOP.The vials were shaken until the mmol/l potassium-phosphate buffer, pH 7.0, with solution was clear. Counting was performed for 4 mmol/l EDTA in a Servall Omnimixerfor 3 min. 5 rnin in a Packard-Tri-Carb liquid scintillation The homogenate was filtered through glass-wool counter. and centrifuged at 10,OOOg for 40 min. The superStandard cyclic AMP assay was performed with
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MATERIAL
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Assayfor Cyclic AMP
a buffer containing 50 mmol/l potassium-phosphate and 2 mmol/l EDTA, pH 7.4, in the following referred to as standard assay buffer. The components were added in the following order: Fifty pl of a solution containing 0.5 pmol [3HH]cyclicAMP in standard assay buffer. Fifty p1 standard or unknown. Hundred pl binding protein solution in standard assay buffer with 1yo(wt/vol) BSA, and Standard assay buffer to a total volume of 350 p.1. In the case of calibration for plasma, cerebrospinal fluid, and amniotic fluid determinations, 50 $1 cyclic-AMP-free material was added before the addition of binding protein. The incubation, separation of free and bound [3H]cyclic AMP, and radioactive counting were performed as described under binding reaction. The counts per minute (c.p.m.) obtained in the presence of unlabeled cyclic AMP (Cx) and c.p.m. in the absence of unlabeled cyclic AMP (C,) were used for the construction of calibration curves by plotting the ratio Cx/Coagainst log concentration in nmol/l of cyclic AMP in the added standard solution. Blood, Cerebrospinal fluid, and amniotic fluid samples were collected by venipuncture, lumbar puncture, and amniocentesis, respectively, and EDTA was added immediately to give a final concentration of 5 mmol/l. The tubes were immediately mixed, centrifuged at 2 "C, and kept at -20 "Cuntil analyzed. Urine samples were frozen at -20 "C as soon as possible after collection. After thawing, before the analysis, the samples were centrifuged if there was any sign of turbidity. Standard assay buffer was added to the desired dilutions. Cyclic-AMP-free plasma, cerebrospinal fluid, and amniotic fluid were prepared from samples without added EDTA (blood samples collected in heparinized tubes). The samples were incubated with 0.03 U phosphodiesterase per ml for 15 h at 32 "C. The phosphodiesterase solution (0.1 5 U/mg protein) had previously been dialyzed against a 50 mmol/l potassium-phosphate buffer, pH 7.4, with 50 mmol/l magnesium sulfate. After incubation, EDTA was added to a final concentration of 5 mmol/l, and the samples were stored at -20 "C. Samples were also pooled for use in the assay.
291
Protein was determined by the method of Lowry et al. (19) and creatinine by the method of Bosnes 8c Taussky (7). Trichloroacetic acid (TCA) extraction was performed by mixing 500 pl of sample and 100 pl 300/, (wtlvol) TCA. After centrifugation, 400 $1 of the supernatant was acidified by adding 45 p1 of 1 mol/l HCl and extracted seven times with three volumes of water-saturated ether. The extract was dried at room temperature for 12 h under airstream and dissolved in standard assay buffer. Ion-exchange column purification of TCAextracted samples and diluted urine (1 :500) was performed on 0.4x3.5 cm AG 1-X8 formate columns, as described by Murad et al. (22). To correct for recovery, 0.01 pmol [3H]cyclic AMP was added to the samples before extraction and purification. The [3H]cyclic AMP used for recovery experiments was purified on an AG 1-X8 formate column before use to remove tritiated water formed during storage.
RESULTS AND DISCUSSlON Preparation of bindinfi protein
As shown in Fig. 1, three distinct peaks of binding activity were found in the eluate from the DEAEcellulose column, tested with both 50 mmol/l sodium acetate, pH 4.0, and 50 mmol/l potassiumphosphate, pH 7.4, as assay buffers. The fractions in peak I were pooled as indicated in Fig. 1, and this material was used in the binding studies mentioned below, and also in the assay system for cyclic AMP. The amount of binding protein obtained was about 80 mg. In the cyclic AMP assays previously reported, the first peak of binding activity from the DEAE-cellulose, eluted by the method of Gilman (13), was used without specification of the number of activity peaks found (11,27). In the work of Gilman (13) the first peak probably contains a mixture of peaks I and I1 in the present work (Fig. 1). The improvement in separation of peaks 1 and I1 was obtained by lowering the pH from 7.0 to 6.8. p H dependence of the cyclic AMP binding As shown in Fig. 2, there was a marked increase in apparent cyclic AMP binding when the pH was increased from 4.0 to 5.0. When the p H was
L. Aksnes & 0.Sovik
292 I\
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40W
Frxtcon no
Fig. 1. DEAE-cellulose chromatography of binding protein. Determination of binding activity in the fractions (15 ml) was carried out as described in the text with 0.5 pmol LH-labeled 3’,5’-monophosphate (cyclic AMP) and 10 p1 of the fractions. A: (O---O)=relative binding activity with 50 mmol/l sodium acetate, pH 4.0, as assay buffer; (---)== potassium phosphate gradient; +--/)=pooled fractions used in the assay. B: (.---.)=relative binding activity with 50 mmol/l potassium phosphate, pH 7.4, as assay buffer; (x-x)=optical density at 280 nm. further increased to 6.2, there was a drop in binding capacity, whereas a clearly improved binding was observed by raising the pH from b.2 to 7.7. Using a potassium-phosphate buffer, there was only a small variation in binding between pH 7 and 8. Somewhat higher binding activity was observed with potassium-phosphate than with Tris-HC1 buffer. For comparison the protein kinase preparation of Gilman (13) and an adrenal protein used by Tsang et al. (ZS), both using acetate buffer, gave maximal binding at pH 4.0 and 5.0, respectively. Nonspecific interference The interference in the standard cyclic AMP assay by CaCl,, MgClZ, NaCl, BSA, plasma, cerebrospinal fluid, amniotic fluid, and diluted
Fig. 2. Effect of pH on adenosine 3’,5’-monophosphate (cyclic AMP) binding activity. Binding was carried out as described in the text using 5 pg binding protein, 0.5 pmol [sH]cyclicAMP, and 50 mmol/l assay buffer, to a total volume of 300 pl. Assay buffers used were (0--O)=sodium acetate acetic acid, (.---.)= potassium phosphate, ( X ---x)=Tris-HCl. urine at different concentrations of unlabeled cyclic AMP is shown in Table I. Albumin up to lo%, NaCl up to 0.5 mol/l, and urine and the divalent cations Ca2+and Mg2+at concentrations up to 5 mmol/l did not interfere at any concentration of unlabeled cyclic AMP. At 7.5 mmol/l and above Ca2f and Mg2+caused a decrease in bound labeled cyclic AMP. The degree of nonspecific interference was dependent on the concentration of unlabeled cyclic AMP in the reaction mixture. This was also observed by Albano et al. (1). Plasma, cerebrospinal fluid, and amniotic fluid caused a small increase in bound labeled cyclic AMP at all concentrations of unlabeled cyclic AMP. Different samples of plasma, cerebrospinal fluid, and amniotic fluid did not show any variation in interference. The same increase was also observed after diluting these samples 1:l with assay buffer. The effect of cyclic GMP, ATP, and AMP on the binding of cyclic AMP is shown in Fig. 3. Cyclic G M P showed the most marked interference, but a concentration about 1 0 0 times that of cyclic AMP was necessary to give the same decrease in [3H]cyclicAMP binding. ATP had no effect at concentrations below 5 >c lop4mol/l, and
Assay for Cyclic AMP
293
Table I. Binding of 3H-labeled adenosine 3‘,5’-monophosphate (cyclic AMP) at different concentrations of unlabeled cyclic AMP made up in different media. Binding studies were performed with 0.5 pmol [3H]cyclic AMP and 50 ~1of the indicated media as described under standard cyclic AMP assay in the text Medium (concentration of cyclic AMP in medium, nmol/l)
[3H]cyclicAMP bound, pmol, rneanfS.D. (n=4) 6
0
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~~
Assay buffer CaCl,, 5 mmol/l CaCl,, 7.5 mmol/l MgClz, 5 mmolil MgCl,, 7.5 mmol/l NaCl, 0.2 mol/l NaCI, 0.5 mol/l BSA, 10% Mixture containing CaCl,, 5 mmol/l; MgCl,, 5 mmol/l; NaCl, 0.2 mol/l; and BSA, lo?; Plasma Cerebrospinal fluid Amniotic fluid Urine (1:lOO)
O3
i
25 ~~
0.272*0.007 0.270+0.008 0.248*0.010 0.268*0.008 0.252+0.008 0.279+0.005 0.273f0.008 0.270f0.004
0.229&0.006 0.231 50.007 0.211&0.007 0.227*0.007 0.209+0.007 0.235+0.007 0.230f0.006 0.227f0.005
0.127f0.003 0.125fO.OO4 0.122&0.006 0.12450.005 0.123*O.OO4 0.12510.003 0.130&0.005 0.130+0.003
0.270&0.006 0.285*0.006 0.282&0.002 0.28410.006 0.268&0.008
0.227&0.005 0.247&0.005 0.243&0.004 0.245f0.005 0.232+0.006
0.130~0.004 0.140&0.003 0.139&0.004 0.137f0.002 0.125+0.003
AMP had no effect below 10V mol/l. In conclusion Ca2’, Mg2+,NaCl, and protein (BSA) had no effect on the cyclic AMP binding within the concentration limits found in extracellular fluids. The specificity of the binding reaction is high, and the cross-reactivity of the binding reaction by cyclic G M P and ATP is no problem in view of the relative levels of cyclic AMP and these nucleotides in biological materials.
AMP
Standard assay conditions
O
,o‘B
U
’ K)-7
a
K)-5
,o.L
nucleotide concentration
-
lo-3
mol
,o-2
/I
Fig. 3. Binding of 3H-labeled 3’,5’-monophosphate (cyclic AMP) in the presence of cyclic AMP, guanoshe 3’,5’-monophosphate (cyclic GMP), adenosine 5’monophosphate (AMP), and adenosine triphosphate (ATP). Binding was carried out as described under standard cyclic AMP assay in the text with 50 p1 of the nucleotide solutions.
Fifty mmol/l potassium phosphate, pH 7.4, was chosen as the standard assay buffer because its p H is very close to that of plasma, cerebrospinal fluid, and amniotic fluid and to the p H where the binding of cyclic AMP isnear maximum. EDTA was added to inhibit the Mg2+-dependent phosphodiesterase present in the samples (3) and to reduce the effect of metal ions on the binding reaction ar,d separation of bound and frec cyclic AMP by charcoal (1, 13). Gilman (13) reported that addition of a protein kinase inhibitor isolated from bovine skeletal muscle increased both the affinity with cyclic AMP and the number of available binding sites on his protein kinase preparation. The same effect was later observed with other protein preparations, among them BSA (21). The effect
294
L. Aksnes & 0.Sovik
t
V
4 Q
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f
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5
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Fig. 4. Double. reciprocal plots of adenosine 3’,5’monophosphate (cyclic AMP) binding. Binding was carried out as described in the text in a total volume of 300 d,using 5 pg binding protein and 50 mmol/l potassium phosphate, pH 7.4, with 2 mmol/l EDTA as assay buffer. (-)=with 1 mg bovine serum albumin (BSA) in the reaction mixture, (O-o)= without BSA.
Fig. 5. Effect of dilution of binding protein on the binding of adenosine 3’,5’-monophosphate (cyclic AMP). Binding was carried out as described under standard cyclic AMP assay in the text with 0.5 pmol [aHlcyclic AMP.
of BSA on the binding properties of the binding protein was investigated also in the present work and was found to activate the binding protein and neutralize the degree of nonspecific effects an the binding due to differences in composition of biological fluids. The optimal effect was obtained by using 1 mg BSA in the reaction mixture (350 ~ 1 ) . The binding, in presence and absence of BSA, was evaluated by double reciprocal plots of bound against free cyclic AMP, as shown in Fig. 4.The association constant was increased from 1.4 to 1.8 x los l/mol, and the number of available sites was also increased. One pg binding protein was found to bind 0.13 and 0.08 pmol cyclic AMP in presence and absence of BSA, respectively. The binding data obtained are the sum of two reactions, the binding of cyclic AMP to the binding protein and the separation of free and bound cyclic AMP. The different methods used for separation of free and bound cyclic AMP are all shown to be more or less influenced by environmental factors. The membrane filtration method is influenced by EDTA, metal ions, and proteins (4, 28, 31). The ion-exchange method is influenced by salts and plasma extracts and gives incomplete uptake of cyclic AMP(28). The charcoal method is influenced
by metal ions (1) and plasma extracts (28), but none of these factors were found to influence the separation in the present study. The amount and grade of charcoal were found to be important factors for satisfactory separation (9). With the amount and grade used in the present study, 99.5% of the free cyclic AMP was absorbed. Maximum binding of [3H]cyclic AMP obtained was 88%. The time elapsed from addition of charcoal to centrifugation was found to be important, probably due to dissociation of the bound cyclic AMP. A nearly linear decrease (about 5%) in bound cyclic AMP was found within the first 10 min. The time factor for removal of supernatant was less important, since no difference was found in specimens taken within the first 10 min after the centrifuge was stopped. The charcoal method is inexpensive and convenient to use for a large number of samples. A saturating concentration of cyclic AMP was chosen because this gave the most precise and sensitive assay with a minimum of interference from other substances. The amount of binding protein chosen was that required to give 50% binding of the 0.5 pmol [“HJcyclicAMP added,
Assay for Cyclic AMP
295
the whole concentration range of cyclic AMP measured. Calibration with a cyclic-AMP-free pool of the same type of fluid that was to be tested was preferred, since this gave an improved reproducibility of the results.
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Plasma sumplts
01
02 0 3
04
05
06
07
08
09
10
W C O
Fig. 6. Typical calibration curve for the assay of adenosine 3’,5’-monophosphate (cyclic AMP). The abscissa shows the ratio of c.p.m. in the presence of unlabeled cyclic AMP (C,) and c.p.m. in the presence of unlabeled cyclic AMP (CO).The ordinate shows the concentration in nmol/l added standard cyclic AMP solution (50 pl) on a logarithmic scale. calculated from a plot shown in Fig. 5. Fig. 6 shows a typical calibration curve. The semilogarithmic plot gave a nearly linear curve between the concentrations 6 and 50 nmol/l. The small nonspecific interference by plasma, cerebrospinal fluid, and amniotic fluid can be corrected for by simple subtraction because the interference was found to be nearly constant for different samples of the same type of fluid over
For the direct determination of cyclic AMP in plasma samples calibration was carried out by addition of 50 pl of a cyclic-AMP-free plasma pool. Addition of cyclic-AMP-free plasma had no effect on the Cx/Co ratio, and between-assay precision was within the level for determinations in assay buffer only (Table 11). The coefficient of variation was less than 7% within the range 6-50 nmol/l for duplicate determinations with six different cyclic-AMP-free plasma samples used fcr calibration. Table I11 shows the validity of the assay. The sample can be diluted 1 : 1 without changing the degree of nonspecific interference, which indicates that the assay conditions neutralize variations in compositions of the various samples. The assay also seems to be free of the reported nonspecific interference caused by the TCA extraction method (1). The immediate addition of EDTA to the samples is important because of the high phosphodiesterase activity, even in frozen samples (3). After addition of EDTA to a final concentration of 5 mmol/l, no decrease in cyclic AMP was observed after 15 h of incubation at 32 “C. Plasma cyclic AMP measured in 10 normal
Table 11. Between-assay reproducibility and precision in the standard adenosine 3’,5’-monophosphate (cyclic AMP) assay for the ratio between c.p.rn. in the presence of unlabeled cyclic AMP (Cx) and c.p.m. in the absence of unlabeled cyclic AMP (C,), and determination of cyclic AMP in nrnol/l. Cyclic AMP standards were assayed on 12 separate days Cyclic AMP, nmol/l
CXICO Cyclic AMP, nmol/l (50 pl) 3 6 12 25 50
100 200
Meanf S.D. (n=12) 0.891&0.020 0.76510.017 0.604&0.018 0.402*0.020 0.225+0.021 0.131 10.015 0.079+0.014
Coefficient of variation (yo) 2.2 2.2
3.0 4.9 9.3 11.5 17.7
S.D. (n-12)
-- 0.3
Coefficient of variation (96)
-- 0.4
9.7 6.8
- 0.7 - 1.8 2.5 Zll.0
-
7.2 5.0 11.0
130.0
15.0
5.6
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Table 111. Direct measurements of adenosine 3',5'-monophosphate (cyclic AMP) in plasma, cerebrospinal fluid (CSF), and amniotic fluid (AF) and measurements of trichloroaceticacid (TCA) extracts and ionexchangepurified extracts of the same fluids. Assay was carried out as described under standard cyclic AMP assay in the text, calibrated with standard assay buffer (+)or cyclic-AMP-free pool of the assayed fluid (++)
pmol cyclic AMP added per ml sample
Preparation
Plasma
CSF
AF
Cyclic-AMP-free Cyclic-AMP-free, diluted 1:1 Cyclic-AMP-free; TCAextracted + Cyclic-AMP-free, ionexchange
10 20 20
19.6f0.9 10.2f0.5 20.1 f1.5 18.8f2.1
20.751.0 9.6f0.7 19.1k2.3 20.5f2.4
19.811.2 10.510.7 19.351.7 19.9f1.9
Collected in EDTA Collected in EDTA, diluted 1:1 -t Collected in EDTA, TCAextracted Collected in EDTA, ionexchange
0 0 0 0
20.3h0.9 10.4f0.5 19.8f1.6 21.0f1.7
22.1f1.4 11.6f0.6 21.4f 1.7 21.3f2.0
30.71 1.4 14.9f0.7 29.1 52.1 31.012.6
20
+ +
++
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Measured cyclic AMP in sample in nmol/l, meanfS.D. (n=4)
+
+ +
+
+
+
young adults (6 females and 4 males) gave a mean of 17.7 nmol/l f 2.5 (S.E.M.)(range, 12.5 to 22.0).
good agreement with reported values in this patient group (18).
Amniotic fluid
Calibration for direct determination in cerebrospinal fluid was performed with 50 pl of a cyclicAMP-free cerebrospinal fluid pool. The betweenassay coefficient of variation was less than 6.5% within the range 6-50 nmol/l for duplicate determinations with six different cerebrospinal fluids used for calibration. The data in Table I11 show good agreement of determinations in undiluted samples, samples diluted 1: 1, samples extracted by TCA, and samples purified on ion-exchange columns. Cerebrospinal fluid samples from 10 patients aged 6-13 years with acute leukemia, taken during routine control of the disease, gave a mean of 21.2 nmo1,'l & 2.0 (S.E.M.) (range, 17.5-24.6), which is in good agreement with values reported elsewhere (23, 25).
For the direct determination of cyclic AMP in amniotic fluid calibration was carried out with the addition of 50 pl of a cyclic-AMP-free amniotic fluid pool. The between-assay coefficient of variation was less than 7.5% within the range of 6 to 50 nmol/l for duplicate determinations with seven different cyclic-AMP-free amniotic fluids used for calibration. The data in Table IIL show good agreement between determinations in undiluted samples, samples diluted 1:1, samples extracted by TCA, and samples purified by ion-exchange chromatography. Although the composition of amniotic fluid changes during pregnancy (l'), samples from different stages of gestation (22-40 weeks) caused no variation in nonspecific interference. The half-life of cyclic AMP in amniotic fluid incubated at 32 "C was 7-10 h, indicating a rapid degradation of the nucleotide. Degradation was also observed in frozen samples (-20 "C). Addition of EDTA prevented the degradation, indicating the presence of a Mg2+-dependent phosphodiesterase. This enzyme activity has not previously been reported in amniotic fluid. A value of 45.6 nmol/lf7.5 (S.E.M.) was found in amniotic fluids from pregnancies complicated by hypertension, which is in
Cerebrospinal fluid
Urine
Diluted urine samples did not bring any problem of nonspecific interference and were consequently assayed directly without addition of cyclic-AMP-free urine for calibration. The urine samples were usually diluted 1 :500. Urine from 20 normal children aged 2-10 years gave a mean value of 5.9 h 1.2 (S.E.M.) nmol cyclic AMP per mg creatinine (range, 2.5- l0.5),
Assay for Cyclic AMP which is in good agreement with values reported by August et al. (2).
ACKNOWLEDGEMENTS This work u a s aided by grants from the Nordic Insulin Fund and from the Naiisen Foundation.
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Received 25 September 1975 Accepted 22 Dwmber 1975
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