Biochemical SocietyTransactions ( 1 99 1 ) 19 347s The
purine
nucleotide
content
in
normal
human
lyl.PhoCyteS
IdARINELLO ENRICO, PAGAN1 ROBERTO, CARLUCCI FILIPPO, MOLINELLI MASSIMO*, VALERIO PATRIZIAO and TABUCCHI ANTONELLA Institute of Biochemistry and Enzymology, *Institute of Pediatrics, OInstitute of Dentistry, University of Siena, Pian dei Mantellini 44, 53100 Siena, Italy. A n important reason for the determination of the purine nucleotide content is that it furnishes information on the behavior of purine nucleotide metabolism, and also on the oxido-reduction state, the energy charge of the cells as well as other parameters. While many comparative determinations are available on the thymocytes, and on different cell lines of the lymphocytes of different species, information regarding the concentration of purine nucleotides in the lymphocytes of human peripheral blood (PBL) is very limited; their values, under normal conditions, are not yet sufficiently standardized. Since this biochemical parameter is very important for an understanding of purine nucleotide metabolism and for physiopathological, diagnostic and comparative purposes, we have evaluated the purine nucleotide levels in the PBL of normal subjects. Blood was withdrawn from 7 normal male, aged between 26 and 58, and fasting since the night before, at 8 a.m. 30 ml of blood was normally sufficient for determination. Lymphocytes were isolated using the Lymphocyte Separation Medium (L.S.M.) from F l o ~ Laboratories (1) and finally suspended (1 x 10 cells/ml) in PBS (phosphate-buffered saline, pH 7). Cells were spun down at 200 x g and deproteinized with HC104; pellets were discarded by centrifugation. Neutralized supernatants were either used immediately or stored at -70°C until HPLC analysis could be performed. The final concentration of HC104 and the time of exposure had a specific effect on subsequent determination and thus concentration and exposure time were staryjardized (we added 200p1 of 0.4 N HC104 to 2 x 10 cells leaving the extract no longer than 15 min at OOC). For HPLC analysis we used a Beckman instrument, equipped with 2 pumps mod.fl0B and an U.V. detector mod.166. A loop1 aliquot of neutralized supernatants was injected for HPLC analysis. The identification of the nucleotides was carried out through retention time and coelution with internal standards. For the separation of all nucleotides we adopted a modification of De Korte's et al. (2) gradient, with a column of Whatman Partisil 10 SAX (250 x 4.6 mm): we used a gradient elution, buffer A (7.5 mM ammonium phosphate, pH 3.8, containing 2% acetonitrile) and buffer B (750 mM ammonium phosphate, pH 4.92, containing 2% acetonitrile). Figure 1 reports a typical chromatogram of the different standards (AMP, GMP, IMP, XMD, NAD, GDP, GTP, ADP and ATP). Linearity was obtained for all amounts of nucleotides used in this study. The minimum amount of these compounds detectable in our mixtures was 1 picomole. Correlation coefficient and the regression equations of the calibration curve were very satisfactory. The overall precision of the retention times and peak areas was confronted with run-to-run and day-to-day precision. The mean of several determinations carried out for the normal subjects is reported in Table 1. The procedure adopted is highly recommendable for an accurate separation and a precise determination of the most important purine nucleotides.
A high dispersion of values can be observed, as shown also by the standard error. This result can be interpreted by the fact that circulating lymphocytes are a mixed population: many of them, in the different subjects, are probably in an "activated form", which involves a different concentration of nucleotides. It is possible that in normal subjects the concentration of nucleotides is always remarkably variable and that a high dispersion of values characterizes the normal cell (3,4,5). 6YP
110 c OoSl
a I
D
9
i
AMP
I
LO
I
I
1
20
10
10
40
SO
60
70
80
MINUTES
Figure 1 HPLC W profile of a standard nucleotide mixture Standard mixture of 9 nucleotides (individual concentration of 2 nmoles. for NAD = 4 nmoles)
Table 1. Nucleotide content of peripheral 1 m hoc tes. Concentrat:onPs inY pmol.106 cells. NAD 1 2 3 4 5 6 7
AMP
IMP GMP
54 / 143 19 1 2 4 6 9 22 3 266 / 1 ll0 6
[email protected] SfiM 33.4
7 54
67 22
2 3 8
3 6 31 58
21.7
49.2
10.6
16.4
XMP
ADP
GDP
ATP
blood
GTP
4422631069194 2587'76ll41xx) 1 2 / 5 4 7 7 5 1 0 2 1 1 8 9 / / 3 l 7 5 5 7 6 8 1 5 0 2 2 2 3 m l l 1 5 8 7 3 3 5 1.1 1.2 585 58 ll67 243 / 52 !B 7 3 1 W 176 8
/
6.1 3.1
ll.7
4.4
540.1 58.7 48.9 8.5
1l19 203.1 9.4 78.6
1. Boyum, A. (1968) Scand.J.Clin.Lab.Invest.Supp1. 97, 31-50 2. De Korte, D.. Haverkort, W.A., Roos, D. & Van Gennip, A.H. (1985) Clin.Chim.Acta 148, 185-196 3. Marijnen, Y.N.T., De Korte, D., Haverkort, W.A., Den Breejen, E.J.S., Van Gennip, A.H. & Ross, D. (1989) Biochim.Biophys.Acta 1012, 148-155 4. Gruber, H.E., Jansen, F., Willis, R.C. & Seegmiller, J.E. (1985) Biochim.Biophys.Acta 846, 135-144 5 . Peters, G.J., De Abreu, R.A. Oosterhof, A. & Veerkamp (1983) Biochim.Biophys.Acta 759, 7-15
This work was financed by a contribution of the Minister0 della Saniti, Istituto Superiore Saniti, Progetto AIDS, 1990, Roma, Italy.
