- Email: [email protected]
The separation and evaluation of puric bases and their nucleosides and nucleotides by thin-layer chromatography
69
Clinica Chimica Acta, 53 (1974) 69-77 0 &evier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6328
THE SEPARATION AND EVALUATION OF PURIC BASES AND THEIR NUCLEOSIDES AND NUCLEOTIDES BY THIN-LAYER CHROMATOGRAPHY
J.J.E.
CHIVOT*,
M.J. LEIBRANDT*,
H.L. ROUVROY*,
J. SIMEON**
and J.P. CAEN**
Laboratoire d’H@‘mostase et de Thrombose Exp@‘rimentale, Institut de Recherches Maladies du Sang, HGpital Saint-Louis, Uniuersite’Paris VII, Paris and Laboratoire CEA, B.P. 61, 92120 Montrouge (France)
(Received
December
sur les Me’dical du
22, 1973)
Summary A method is described of thin-layer chromatography and liquid scintillation suitable for the separation and the evaluation of nucleotides, nucleosides and puric bases. The procedure employs PEI-cellulose plates with migration in two dimensions. The identification of the separated constituents utilizes radioactivity of ’ 4 C or UV light. The radioactive compounds are evaluated by liquid scintillation counting. This technique offers ease of manipulation, use of small quantities of material, highly consistent and reproducible results and good separation of the individual compounds. An application of this technique to a plasma system is proposed. This method enables a present study to be made of the metabolism of platelets in our laboratory.
Platelet adenine nucleotide metabolism is closely related to its essential function which is mainly aggregation. In view of the important part of adenosine [l] and degradation products [2] in the mechanism of inhibition of platelet aggregation, we previously studied the behaviour of these metabolites in the plasma and described a separative method using paper chromatography [3] . This technique was applied to the separation of a mixture of radioactive bases and nucleosides at a concentration less than 10e6 M.
* Present address: Service de Chimie Clinique, Hbpital de Nanterre. 92014 Nanterre, France. ** Present address: Laboratoire d’He’mostase et de Thrombose Expdrimentale. ERA 335 CNRS, stitut de Recherches SUTles Maladies du Sang, HBpital Saint-Louis, 75010 Paris, France.
In-
70
Due to interest not only in the metabolism of adenosine in plasma but also in different mammalian platelets [4], it was necessary to find a more suitable technique for obtaining a better separation of bases, nucleosides and nucleotides. This technique is described in this paper and it involves analysis on cellulose plates subjected to two-dimensional thin-layer chromatography. An application for the separation of bases and nucleosides obtained from plateletpoor plasma with [8- ’ 4 C] adenosine is described, in addition to the following procedures: (1) The methods chosen for the separation of adenosine, adenine, inosine, hypoxanthine, adenosine and inosine monophosphates (AMP and IMP), adenosine and inosine diphosphates (ADP and IDP), adenosine and inosine t~phosphates (ATP and ITP) and cyclic adenosine monophosphate (cyclic AMP) by thin-layer chromato~aphy; (2) the identification of the separated constituents by autoradiog~phy and UV light; (3) the evaluation of the radioactivity associated with each isolated constituent; and (4) an application of this technique to a biological system. Materials Radioactive chemicals [8-l 4 C] Adenosine
5’-monophosphate (ammonium salt), spec. act. 47.7 mCi/ml; [8-’ 4 C] adenosine 3,5monophosphate (ammonium salt), spec. act. 53 mCi/mmole; [8-r 4 C] adenosine 5’-triphosphate (ammonium salt), spec. act. 26 spec. act. 50 mCi/mmole; [8-’ 4 C] adenine mCi/mmole; 6-l 8- ’ 4 C] adenosine, (hydrochloride), spec. act. 45 mCi/mmole; [S-l 4 C] hypox~thine (hydrochloride), spec. act. 47.4 mCi/mmole; and [8-l 4 C] inosine, spec. act. 48 mCi/ mmole, were supplied by Le Commissariat 1 1’Energie Atomique (CEA), (ammonium salt), spec. act. 52 mCi/ France. [8-l 4 C] Adenosine 5’-diphosphate mmole was obtained from the Radiochemical Center, Amersham, Bucks., England. Thin-layer
chromatography
PEI-cellulose 0.1 cm.
Plates
TLC-Ready,
Plastic
Sheets
F 1440,
Estar) were supplied
by Kodak,
20 cm X 20 cm X
Au toradiography
Kodirex Platelet-poor
films (support
France.
plasma
Blood from normal human healthy donors and from Wistar rats was collected using 3.8% sodium citrate as an anticoagulant (1 part anticoagulant to 9 parts blood). The blood was centrifuged for 20 min at 25000 X g to obtain platelet-poor plasma (PPP). Methods separation of ~hromato~aphy.
the
The following
samples
to
be analysed
samples were prepared
by
two-dimensional
for analysis.
thin-layer
71
(1) A mixture of l 4 C-labelled compounds, containing adenosine, adenine, i;iosine, hypoxanthine, AMP, ADP, ATP, cyclic AMP and the non-radioactive IMP, IDP, ITP compounds. The solution was diluted to a concentration of 10m4 M and 5 &i/ml activity, with respect to each labelled compound and lo-’ M with respect to each non-radioactive compound using sodium phosphate buffer 0.1 M, pH 7.4. (2) For the evaluation of the fate of [8-l 4 C] adenosine in human plateletpoor plasma (PPP): 1.8 ml PPP + 0.2 ml [a-’ 4 C] adenosine 20 pCi/ml pH 7.4 were incubated at 37”. For rat PPP, the mixture contained 0.9 ml PPP and 0.1 ml [8-’ 4 C] adenosine 20 &i/ml. The plasmas were deproteinised by the addition of 2 ml 96% ethanol and stored at -2” for 30 min, following by centrifugation at 27 000 X g at 4” for 20 min. The supernatant was removed and stored at -40” until analysed. Application of the sample to the plate Each sample was applied to the lower left corner of the plate at a point 2 cm from the horizontal and vertical edges. On each plate were also spotted
Fig. 1. Radiochromatogram solvent. System number 1.
of a mixture
of 14C-labelled
bases. nucleosides
and nucleotides
separated
by
72
two mixtures of the known products (10m5 I.rmole of each radioactive compound and 10m3 @mole of each inactive compound from a concentrated mixture of the known products): the first was to control development in the first dimension, and was placed 2 cm from the lower horizontal and right vertical edges of the plate, and the second, to control development in the second dimension, was applied 2 cm from the top horizontal and left vertical edges (Fig. 1). Solvent
systems
System no.1. 1st dimension: tri-distilled water; migration front of 10 cm on PEI-cellulose [ 51. 2nd dimension: isopropanol~atura~d (NH4 )Z SO4 solution-bi~distilled water (2:79:19, by vol.) pH 4.7; migration front of 10 cm in 1 h 15 min on PEI-cellulose [6]. System no.2 1st dimension: this was carried out in 3 different development tanks: (a) 1st tank: 0.2 M LiCl, developed for 2 min; (b) 2nd tank: 1 M LiCl, developed for 6 min; (c) 3rd tank: 1.6 M solvent allowed to migrate to 13 cm which required 2 h 30 min on PET-cellulose. The plates were “rinsed” in pure dry methanol for 15 min, to eliminate the LiCl as well as the bases and nucleotides. 2nd dimension: this was also carried out with 3 different development tanks using sodium formate-formic acid buffer, pH 3.5. (a) 1st tank: 0.5 M for 30 s; (b) 2nd tank: 2 M for 2 min; (c) 3rd tank: 4 M, solvent allowed to migrate to 15 cm which required 1 h 30 min. ~~a~e~ent
of the separated constituents on the developed plate The identification of the separated constituents was achieved by autoradiography as described by Chivot et al. [3], The duration of contact of the developed plate and film was between 3 and 6 days. The identification of the inosine nucleotides was achieved by UV spectroscopy at 254 nm.
The identified regions of radioactivity were removed from the plate in the form of small p~~lelo~~s of 2 cm X 2.5 cm and placed in polyethylene counting vials. Each sample was treated with 1.5 ml of distilled water and followed by the addition of 15 ml of Insta-Gel. After agitation, a gel was obtained containing in suspension the fragment of the plastic support from the plate, which is transparent to the photons. The samples were counted in a refrigerated scintillation counter after they had been equilibrated to the temperature of the machine. Evaluation and allowance for the quenching was made and the corrected results were obtained via a teletype expressed in dpm and in nCi. Results Table I iIlustrates the average values of the RF values for investigated compounds using both systems 1 and 2. There was some slight v~iability in these quoted values according to the nature of the samples to be analysed,
73 TABLE
I
AVERAGE AND
RF
VALUES
FOR
INVESTiGATED
COMPOUNDS
ON
PEI-CELLULOSE
SCWLEICHER
SCHULL
System
system
1
1st dimension
2nd
(10
(10
cm)
dimensian cm)
2
--
1st dimension
2nd
(13
(15
cm)
dimension cm)
Non-radioactive
IMP
0.65
0.66
0.54
0.58
0.63
0.65
Non-radioactive
IDP
0.72
0.70
0.48
0.48
0.43
0.45
Non-radioactive
ITP
0.77
0.74
0.38
0.35
0.22
0.23
0.09
0.08
0.48
0.45
0.57
0.62
AMP
0.28
0.27
0.46
0.42
0.65
0.62
ADP
0.30
0.29
0.40
0.38
0.47
O.&O
[14C1
ATP
0.34
0.31
0.30
0.30
0.23
0.25
[14C1
Inosine
0.73
0.69
0.42
0.41
0.69
0.72
0.71
0.14
[14Cl
Hypoxanthine
0.47
0.47
0.25
0.25
0.48
0.45
0.54
0.57
[14Cl
Adenosine
0,52
0.51
0.12
0.12
0.46
0.44
0.61
0.63
1 14Cl
Adenine
0.34
0.39
0.08
0.08
0.22
0.22
0.52
0.54
Lg4Cl
lab&&d
f%I 1’%f
cyclic
AMP
which justified the inclusion of marginal control samples each time for compariSOn.
A deposit of 3 to 5 ~1, according to the activity of radioactive molecules, was found to be the smahest volume that could be applied to a plate and allowed an easy interpretation of the separated products by autoradiography. The best and most consistent results were obtained when material was applied to the plate in l-p1 aliquots. The reproducibility of the l-p1 micropipets was tested by spotting 8 X l-p1 samples of /3-[8-” 4 C] adenosine of known concentration, developing the plates and isolating the product and then counting the radioactivity associated with the isolated spots. The observed variation in cpm associated with the isolated spots was not superior to that of the statistical fluctuation, which was 2 m, where iV is the average of the total cpm associated with the 8 spotted samples; e.g. if N = 2500, the statistical variation will be + 100. We have compared various fluids for their efficiency in counting samples
TABLE
II
SCINTILLATION
FLUID
EFFICIENCY
FOR
MEASURING
_~_._. Nature
of scintillation
fluid
20 ml Kinard 0.5 15 1.5
ml water
ml Kinard
ml Insta-Gel ml water
Efficiency
48.3 + 20
74 47.4
* 15 ml InSta-Gel
76.6
3 ml water
•t 15 ml In?&-Gel
71.2
6 ml water
f
64.9
15 ml In&a-Gel
fi-[8-‘4Cl ~_~.
(W)
ADENOSINE
RADIOACTIVITY -.
of known specific activity as shown in Table II. In&a-Gel presents fewer technical problems than Kinard [3]. The best results were obtained when 1.5 ml of water was added to 15 ml of In&a-Gel which markedly raised the sensitivity of the method. Fig. 1 shows the separation obtained from a mixture of ’ 4 C-labelled bases, nucleosides and nucleotides using solvent system No.1. Bases, nucleosides and cyclic AMP are well separated, while nucleotides are distributed in two groups, namely adenine nucleotides and inosine nucleotides, On the other hand, Fig. 2 shows that the nucleotides are separated after the elimination of bases and nucleosides by elution with the dry methanol treatment. Fig, 3 reveals the sep~tion obtained from the analysis of human plateletpoor plasma (PPP). We observed the degradation of adenosine in inosine by the adenosine deaminase, aminohydrol~e, which catalyses the deamination of adenosine. Fig. 4 shows the ’ 4 C-labelled compounds from rat plasma. We observed a decrease of adenosine and an appearance of inosine and nucleotides.
Fig. 2. Radiochromatogram solvent. Sysdem number 2.
of a mixture
of 1 4C-labelled
bases, nucfeosides and nucleotides
separated
by
75
Fig. 3. Radiocbromatogram of metabolites obtained after 20 min incubation: 1.8 ml human PPP and 0.2 ml @-[S-l 4 C] adenosine (20 pCi/ml) and ethanol deproteinisation. System number 1.
Discussion Several methods have been described for separation of bases, nucleosides and nucleotides [3,7-93 in platelet-poor and platelet-rich plasma. Whether these methods use paper [ 31 or thin-layer chromatography [‘I--9], they do not allow a good separation of all reaction products. This method using paper chromatography is not only time consuming and complicated but often not reproducible [3]. Murakami and Odake [lo] have described a technique of thin-layer chromatography after separation of all the nucleotides by column chromatography, requiring large quantities of material. In order to avoid degradation of ADP and mainly ATP by acid or alkaline solvent systems [U-18], we have chosen a bidimensional system in which the different solvents were approximately neutral. This technique also allows a simultaneous separation of guanine compounds, cyclic GMP and cyclic adenosine 3,5’-monophosphate. It can, therefore, be applied to assess the levels of these two compounds in platelets and in biological samples in general.
Fig. 4. Radiochromatogram of metabolites obtained after 20 min incubation: 0.9 ml rat PPP + 0.1 ml /3-18-14Cladenosine (20 &CilmlI and ethanol deproteinisation. System number 1.
Furthermore the method described in this paper, in which the plastic plate was cut into small pieces and put into tubes for the radioactivity counting at an efficiency above 75%, is a more rapid and reproducible one than scraping with the scalpel to remove the labelled spots. This technique can also be used for de~rmjning the plasmatic adenosine deaminase activity [19]. Acknowledgement This work was supported by French Atomic Energy Commission Grant 2003 R, INSERT Grant ATP 71-2405-l. References 1 2 3 4
G.V.R. Born, Nature, 194 (1962) 927 J.P. Caen, C.S.P. Jenkins, H. Michel and R. Bellanger, Nature. 239 (2972) 211 J.J. Chivot, D. Depernet and J.P. Caeo, Cfin. Chim, Acts, 27 (1970) 241 C.S.P. Jenkins, J.P. Caen, H. Vainer and J-P. Pokutecky, Nature. 239 (1972) 210
5 J.E. Ciardi and E.P. Anderson. Analyt. Biochem.. 6 K. Randerath and E. Randerath, J. Chromatogr., 7 8 9 10 11 12 13 14 15 16 17 18 19
22 (1968) 22 (1966)
398 110
D.M. Ireland, Biochem. J., 105 (1967) 857 H. Holmsen and M.C. Rosenberg. Biochim. Biophys. Acta, 155 (1968) 326 K. Odake and J.L. Ambrus, Amer. J. Pathol., 56 (1969) 393 M. Murakami and K. Odake. Thrombosis Diathes. Haemorrh.. 25 (1971) 223 K. Randerath and E. Randerath, J. Chromatogr.. 16 (1964) 111 E. Randerath and K. Randerath, J. Chromatogr., 16 (1964) 126 G. Pataki and A. Niederwieser. J. Chromatogr.. 29 (1967) 133 G. Pataki, In J.C. Giddings and R.A. Keller (Eds), Advances in Chromatography, Vol. 7, Marcel Dekker, New York, 1968 A. Niederwieser and CC. Honegger, In J.C. Giddings and R.A. Keller (Eds), Advances in Chromatography, Vol. 2. Ch. 4, M. Dekker. Inc. New York, 1966 A.P. Remenchik and J. Bernsohn. Analyt. Biochem., 18 (1967) 1 P.J. Orsulak, W. Haab and M.D. Appleton, Analyt. Biochem., 23 (1968) 156 H.P. Raaen and F.E. Kraus, J. Chromatogr.. 35 (1968) 531 J.P. Caen, C.S.P. Jenkins, H. Michel, J.J. Chivot. S. Levy-Toledano, F. Rendu, Series Haematologica, 3 (1973) 317
Clinica Chimica Acta, 53 (1974) 69-77 0 &evier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6328
THE SEPARATION AND EVALUATION OF PURIC BASES AND THEIR NUCLEOSIDES AND NUCLEOTIDES BY THIN-LAYER CHROMATOGRAPHY
J.J.E.
CHIVOT*,
M.J. LEIBRANDT*,
H.L. ROUVROY*,
J. SIMEON**
and J.P. CAEN**
Laboratoire d’H@‘mostase et de Thrombose Exp@‘rimentale, Institut de Recherches Maladies du Sang, HGpital Saint-Louis, Uniuersite’Paris VII, Paris and Laboratoire CEA, B.P. 61, 92120 Montrouge (France)
(Received
December
sur les Me’dical du
22, 1973)
Summary A method is described of thin-layer chromatography and liquid scintillation suitable for the separation and the evaluation of nucleotides, nucleosides and puric bases. The procedure employs PEI-cellulose plates with migration in two dimensions. The identification of the separated constituents utilizes radioactivity of ’ 4 C or UV light. The radioactive compounds are evaluated by liquid scintillation counting. This technique offers ease of manipulation, use of small quantities of material, highly consistent and reproducible results and good separation of the individual compounds. An application of this technique to a plasma system is proposed. This method enables a present study to be made of the metabolism of platelets in our laboratory.
Platelet adenine nucleotide metabolism is closely related to its essential function which is mainly aggregation. In view of the important part of adenosine [l] and degradation products [2] in the mechanism of inhibition of platelet aggregation, we previously studied the behaviour of these metabolites in the plasma and described a separative method using paper chromatography [3] . This technique was applied to the separation of a mixture of radioactive bases and nucleosides at a concentration less than 10e6 M.
* Present address: Service de Chimie Clinique, Hbpital de Nanterre. 92014 Nanterre, France. ** Present address: Laboratoire d’He’mostase et de Thrombose Expdrimentale. ERA 335 CNRS, stitut de Recherches SUTles Maladies du Sang, HBpital Saint-Louis, 75010 Paris, France.
In-
70
Due to interest not only in the metabolism of adenosine in plasma but also in different mammalian platelets [4], it was necessary to find a more suitable technique for obtaining a better separation of bases, nucleosides and nucleotides. This technique is described in this paper and it involves analysis on cellulose plates subjected to two-dimensional thin-layer chromatography. An application for the separation of bases and nucleosides obtained from plateletpoor plasma with [8- ’ 4 C] adenosine is described, in addition to the following procedures: (1) The methods chosen for the separation of adenosine, adenine, inosine, hypoxanthine, adenosine and inosine monophosphates (AMP and IMP), adenosine and inosine diphosphates (ADP and IDP), adenosine and inosine t~phosphates (ATP and ITP) and cyclic adenosine monophosphate (cyclic AMP) by thin-layer chromato~aphy; (2) the identification of the separated constituents by autoradiog~phy and UV light; (3) the evaluation of the radioactivity associated with each isolated constituent; and (4) an application of this technique to a biological system. Materials Radioactive chemicals [8-l 4 C] Adenosine
5’-monophosphate (ammonium salt), spec. act. 47.7 mCi/ml; [8-’ 4 C] adenosine 3,5monophosphate (ammonium salt), spec. act. 53 mCi/mmole; [8-r 4 C] adenosine 5’-triphosphate (ammonium salt), spec. act. 26 spec. act. 50 mCi/mmole; [8-’ 4 C] adenine mCi/mmole; 6-l 8- ’ 4 C] adenosine, (hydrochloride), spec. act. 45 mCi/mmole; [S-l 4 C] hypox~thine (hydrochloride), spec. act. 47.4 mCi/mmole; and [8-l 4 C] inosine, spec. act. 48 mCi/ mmole, were supplied by Le Commissariat 1 1’Energie Atomique (CEA), (ammonium salt), spec. act. 52 mCi/ France. [8-l 4 C] Adenosine 5’-diphosphate mmole was obtained from the Radiochemical Center, Amersham, Bucks., England. Thin-layer
chromatography
PEI-cellulose 0.1 cm.
Plates
TLC-Ready,
Plastic
Sheets
F 1440,
Estar) were supplied
by Kodak,
20 cm X 20 cm X
Au toradiography
Kodirex Platelet-poor
films (support
France.
plasma
Blood from normal human healthy donors and from Wistar rats was collected using 3.8% sodium citrate as an anticoagulant (1 part anticoagulant to 9 parts blood). The blood was centrifuged for 20 min at 25000 X g to obtain platelet-poor plasma (PPP). Methods separation of ~hromato~aphy.
the
The following
samples
to
be analysed
samples were prepared
by
two-dimensional
for analysis.
thin-layer
71
(1) A mixture of l 4 C-labelled compounds, containing adenosine, adenine, i;iosine, hypoxanthine, AMP, ADP, ATP, cyclic AMP and the non-radioactive IMP, IDP, ITP compounds. The solution was diluted to a concentration of 10m4 M and 5 &i/ml activity, with respect to each labelled compound and lo-’ M with respect to each non-radioactive compound using sodium phosphate buffer 0.1 M, pH 7.4. (2) For the evaluation of the fate of [8-l 4 C] adenosine in human plateletpoor plasma (PPP): 1.8 ml PPP + 0.2 ml [a-’ 4 C] adenosine 20 pCi/ml pH 7.4 were incubated at 37”. For rat PPP, the mixture contained 0.9 ml PPP and 0.1 ml [8-’ 4 C] adenosine 20 &i/ml. The plasmas were deproteinised by the addition of 2 ml 96% ethanol and stored at -2” for 30 min, following by centrifugation at 27 000 X g at 4” for 20 min. The supernatant was removed and stored at -40” until analysed. Application of the sample to the plate Each sample was applied to the lower left corner of the plate at a point 2 cm from the horizontal and vertical edges. On each plate were also spotted
Fig. 1. Radiochromatogram solvent. System number 1.
of a mixture
of 14C-labelled
bases. nucleosides
and nucleotides
separated
by
72
two mixtures of the known products (10m5 I.rmole of each radioactive compound and 10m3 @mole of each inactive compound from a concentrated mixture of the known products): the first was to control development in the first dimension, and was placed 2 cm from the lower horizontal and right vertical edges of the plate, and the second, to control development in the second dimension, was applied 2 cm from the top horizontal and left vertical edges (Fig. 1). Solvent
systems
System no.1. 1st dimension: tri-distilled water; migration front of 10 cm on PEI-cellulose [ 51. 2nd dimension: isopropanol~atura~d (NH4 )Z SO4 solution-bi~distilled water (2:79:19, by vol.) pH 4.7; migration front of 10 cm in 1 h 15 min on PEI-cellulose [6]. System no.2 1st dimension: this was carried out in 3 different development tanks: (a) 1st tank: 0.2 M LiCl, developed for 2 min; (b) 2nd tank: 1 M LiCl, developed for 6 min; (c) 3rd tank: 1.6 M solvent allowed to migrate to 13 cm which required 2 h 30 min on PET-cellulose. The plates were “rinsed” in pure dry methanol for 15 min, to eliminate the LiCl as well as the bases and nucleotides. 2nd dimension: this was also carried out with 3 different development tanks using sodium formate-formic acid buffer, pH 3.5. (a) 1st tank: 0.5 M for 30 s; (b) 2nd tank: 2 M for 2 min; (c) 3rd tank: 4 M, solvent allowed to migrate to 15 cm which required 1 h 30 min. ~~a~e~ent
of the separated constituents on the developed plate The identification of the separated constituents was achieved by autoradiography as described by Chivot et al. [3], The duration of contact of the developed plate and film was between 3 and 6 days. The identification of the inosine nucleotides was achieved by UV spectroscopy at 254 nm.
The identified regions of radioactivity were removed from the plate in the form of small p~~lelo~~s of 2 cm X 2.5 cm and placed in polyethylene counting vials. Each sample was treated with 1.5 ml of distilled water and followed by the addition of 15 ml of Insta-Gel. After agitation, a gel was obtained containing in suspension the fragment of the plastic support from the plate, which is transparent to the photons. The samples were counted in a refrigerated scintillation counter after they had been equilibrated to the temperature of the machine. Evaluation and allowance for the quenching was made and the corrected results were obtained via a teletype expressed in dpm and in nCi. Results Table I iIlustrates the average values of the RF values for investigated compounds using both systems 1 and 2. There was some slight v~iability in these quoted values according to the nature of the samples to be analysed,
73 TABLE
I
AVERAGE AND
RF
VALUES
FOR
INVESTiGATED
COMPOUNDS
ON
PEI-CELLULOSE
SCWLEICHER
SCHULL
System
system
1
1st dimension
2nd
(10
(10
cm)
dimensian cm)
2
--
1st dimension
2nd
(13
(15
cm)
dimension cm)
Non-radioactive
IMP
0.65
0.66
0.54
0.58
0.63
0.65
Non-radioactive
IDP
0.72
0.70
0.48
0.48
0.43
0.45
Non-radioactive
ITP
0.77
0.74
0.38
0.35
0.22
0.23
0.09
0.08
0.48
0.45
0.57
0.62
AMP
0.28
0.27
0.46
0.42
0.65
0.62
ADP
0.30
0.29
0.40
0.38
0.47
O.&O
[14C1
ATP
0.34
0.31
0.30
0.30
0.23
0.25
[14C1
Inosine
0.73
0.69
0.42
0.41
0.69
0.72
0.71
0.14
[14Cl
Hypoxanthine
0.47
0.47
0.25
0.25
0.48
0.45
0.54
0.57
[14Cl
Adenosine
0,52
0.51
0.12
0.12
0.46
0.44
0.61
0.63
1 14Cl
Adenine
0.34
0.39
0.08
0.08
0.22
0.22
0.52
0.54
Lg4Cl
lab&&d
f%I 1’%f
cyclic
AMP
which justified the inclusion of marginal control samples each time for compariSOn.
A deposit of 3 to 5 ~1, according to the activity of radioactive molecules, was found to be the smahest volume that could be applied to a plate and allowed an easy interpretation of the separated products by autoradiography. The best and most consistent results were obtained when material was applied to the plate in l-p1 aliquots. The reproducibility of the l-p1 micropipets was tested by spotting 8 X l-p1 samples of /3-[8-” 4 C] adenosine of known concentration, developing the plates and isolating the product and then counting the radioactivity associated with the isolated spots. The observed variation in cpm associated with the isolated spots was not superior to that of the statistical fluctuation, which was 2 m, where iV is the average of the total cpm associated with the 8 spotted samples; e.g. if N = 2500, the statistical variation will be + 100. We have compared various fluids for their efficiency in counting samples
TABLE
II
SCINTILLATION
FLUID
EFFICIENCY
FOR
MEASURING
_~_._. Nature
of scintillation
fluid
20 ml Kinard 0.5 15 1.5
ml water
ml Kinard
ml Insta-Gel ml water
Efficiency
48.3 + 20
74 47.4
* 15 ml InSta-Gel
76.6
3 ml water
•t 15 ml In?&-Gel
71.2
6 ml water
f
64.9
15 ml In&a-Gel
fi-[8-‘4Cl ~_~.
(W)
ADENOSINE
RADIOACTIVITY -.
of known specific activity as shown in Table II. In&a-Gel presents fewer technical problems than Kinard [3]. The best results were obtained when 1.5 ml of water was added to 15 ml of In&a-Gel which markedly raised the sensitivity of the method. Fig. 1 shows the separation obtained from a mixture of ’ 4 C-labelled bases, nucleosides and nucleotides using solvent system No.1. Bases, nucleosides and cyclic AMP are well separated, while nucleotides are distributed in two groups, namely adenine nucleotides and inosine nucleotides, On the other hand, Fig. 2 shows that the nucleotides are separated after the elimination of bases and nucleosides by elution with the dry methanol treatment. Fig, 3 reveals the sep~tion obtained from the analysis of human plateletpoor plasma (PPP). We observed the degradation of adenosine in inosine by the adenosine deaminase, aminohydrol~e, which catalyses the deamination of adenosine. Fig. 4 shows the ’ 4 C-labelled compounds from rat plasma. We observed a decrease of adenosine and an appearance of inosine and nucleotides.
Fig. 2. Radiochromatogram solvent. Sysdem number 2.
of a mixture
of 1 4C-labelled
bases, nucfeosides and nucleotides
separated
by
75
Fig. 3. Radiocbromatogram of metabolites obtained after 20 min incubation: 1.8 ml human PPP and 0.2 ml @-[S-l 4 C] adenosine (20 pCi/ml) and ethanol deproteinisation. System number 1.
Discussion Several methods have been described for separation of bases, nucleosides and nucleotides [3,7-93 in platelet-poor and platelet-rich plasma. Whether these methods use paper [ 31 or thin-layer chromatography [‘I--9], they do not allow a good separation of all reaction products. This method using paper chromatography is not only time consuming and complicated but often not reproducible [3]. Murakami and Odake [lo] have described a technique of thin-layer chromatography after separation of all the nucleotides by column chromatography, requiring large quantities of material. In order to avoid degradation of ADP and mainly ATP by acid or alkaline solvent systems [U-18], we have chosen a bidimensional system in which the different solvents were approximately neutral. This technique also allows a simultaneous separation of guanine compounds, cyclic GMP and cyclic adenosine 3,5’-monophosphate. It can, therefore, be applied to assess the levels of these two compounds in platelets and in biological samples in general.
Fig. 4. Radiochromatogram of metabolites obtained after 20 min incubation: 0.9 ml rat PPP + 0.1 ml /3-18-14Cladenosine (20 &CilmlI and ethanol deproteinisation. System number 1.
Furthermore the method described in this paper, in which the plastic plate was cut into small pieces and put into tubes for the radioactivity counting at an efficiency above 75%, is a more rapid and reproducible one than scraping with the scalpel to remove the labelled spots. This technique can also be used for de~rmjning the plasmatic adenosine deaminase activity [19]. Acknowledgement This work was supported by French Atomic Energy Commission Grant 2003 R, INSERT Grant ATP 71-2405-l. References 1 2 3 4
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