378s Biochemical SocietvTransactions ( 1 992) 20 Purine nucleotide, nucleoaide and nucleobase content in human lymphocytes from normal subjects and AIDS patients
oxidase activity. *UP, 0 '
lPDx
CARLUCCI F., TABUCCHI A., CONSOLNAGNO E., RE M.C.*, mlNARI P.*, MARINELLO E., PAGAN1 R. Institute of Biochemistry and Enzymology University of Siena - Italy Institute of Microbiology - University of Bologna During previous experiments on the v i n e nucleotide content of human lymphocytes from healthy subjects and HIV-seropositive patients, we described a peculiar pattern and interesting variations in the values of NAD, IMP, GHP, ADP, GDP, ATP, GTP C1-41. In order to establish the metabolic reasons for these changes, we evaluated nucleosides and nucleobasea aa a measure of catabolic rate. A simple method, based on fast ion-pair reversed-phase HPU: was taken from the literature C51, it permits complete and simultaneous separation of purine nucleotides, nucleoaides and nucleobases in only 20 min. Here we report the preliminary data on nucleoside and nucleobase content in human peripheral (PBL) deduced from these blood lymphocytes determinations. For the preparation of lymphocytes, blood was taken at 8 am after overnight fasting. 10 ml of heparinized blood from healthy subjects was usually sufficient for the determination. The lymphocytes were prepared as already reported [l-31, and the pellet was resuspended with ice-cold 0 . 4 N perchloric acid C1-31. For the chromatographic determinations we used a Beckman System Gold Apparatus, equipped with a variable wevelength detector, set at 254 nm, and a prepacked 3 um Beckman C18 column ( 7 . 5 cm x 4.6 m i.d.). The chromatographic conditions are reported in
Table 1. Table 1. Chromatographic conditions. BUFFERS: (A) Low-ionic strength
(B) Low-ionic strength
GRAD1ENT :
FIMJ RATE:
100 mM potassium dihydrogen phosphate pH 6.0 8 mM tetrabutylanrmonium hydrogen sulphate 100 mM potassium dihydrogen phosphate pH 6.0 8 mM tetrabutylammonium hydrogen sulphate 30 X (v/v) methanol 2.5 2.5 5.0 3.0
min min min min 5.0 min 4 . 0 min
A B B B 100 X B down to 0 % B 100 X up to 20 % up to 40 X up to 100 X
1.5 ml/min
The analyses were performed at room temperature. 1 . 5 x 10'3 cells was injected, overloading the injection loop with 50 u l . Peak identities were confirmed by coelution with internal standards and detection at different wavelengths. The pattern of a standard mixture is reported in Fig.1. In addition to purine nucleotides, nucleosides and nucleobases, the oxidized and reduced pyridine coenzymes were also separated, but, obviously, in our FCA extract NADPH and NADH were not detectable since they are easily oxidized. The xanthine peak was also absent because lymphocytes do not exhibit xanthine 20 u1 of extract, corresponding to
0
I
0
"
m
minuks
Fig.1. Ion-pair reversed-phase HPU: of a standard mixture using the chromatographic conditions reported in Table 1. Table 2. Levels of W i n e nucleotides, nucleoaides and nucleobaaes in PBL from healthy subjects and AIDS patientb. Values, given as p~ol/lO'j cells, are the mean + S.E. of 4 determinations. METABOLITE: SUBJECE
A Hypoxanthine Inosine aUanosine
IMP NAD Adenosine
AMP GDP
NADP ADP GTP ATP
115 .L 85 i 21k 85 t 75 k 19* 63 f 88 f 3 t 587 f 220 i 990 i
B 41 21
8 17 15 7 25 22
1 126
30 93
C
95 f 13 90 f 17 49 f 11 41 f 6 1 2 ~ 5 14f 2 48 f 10 92 f 21 32 i 6 36 k 8 1 6 f 5 20;t 3 30 f 18 46 f 21 49 f 15 179 L 38 7;t 3 20* 7 74 i 5 579 f 160 109 f 43 355 ;t 70 776 i 78 1431 f 137
SUBJECTS: A-healthy; 8-asymptomatic;
C-symptomatic
The purine nucleotide, nucleoside and nucleobaae content of peripheral blood lymphocytes from healthy subjects and AIDS patients is reported in Table 2. The HPU: procedure adopted allows the simultaneous evaluation of most purine compounds, giving more and better information than other methods tested to date, which require comparative analysis. The results obtained in AIDS subjects confirm the already reported data [l-41 on nucleotide levels, except for NAD IMP and ADP. Nucleosidea content did not vary significantly in sick patients (except inosine which decreased), neither did hypxanthine levels change. Consequently the generally increased nucleotide content occurring during the infection, might be related to accelerated synthesis. This work was financed by the Italian Secretariat for Health, National Institute of Health, IV AIDS Project, Rome, Italy. References I. Tabucchi, A., Carlucci, F., Ramazzotti. E., Re, M.C., Marinello, E., Rubino, M., Pagani, R. (1991) Biomed. & Pharmacother. 45, 25-29. 2 . Carlucci, F., Tabucchi, A., Consolmagno, E., Tagliaferri, P., Porcelli, B., Marinello, E.. Leoncini, R., Pagani, R. (1992) Biomed. & Phamnacother. 46, 7-12. 3. Tabucchi, A., Carlucci, F., Rubino. M., Marinello, E., Ramazzotti, E., Re. M.C., Pagani, R., (1991) Giorn.It.Chim.Clin. 16, 231-237. 4. Tabucchi, A., Carlucci, F., Marinello, E., Re, M.C., Bisozzi, L., Cacopardo, F., Pagani, R. (1991) Rec.Progr.Med. 82, 581-584. 5. Stocchi, V., Cucchiarini, L., Canestrari, P., Piacentini, M.P., Fornaini, G . (1987) Anal.Biochem. 167. 181-190.
oxidase activity. *UP, 0 '
lPDx
CARLUCCI F., TABUCCHI A., CONSOLNAGNO E., RE M.C.*, mlNARI P.*, MARINELLO E., PAGAN1 R. Institute of Biochemistry and Enzymology University of Siena - Italy Institute of Microbiology - University of Bologna During previous experiments on the v i n e nucleotide content of human lymphocytes from healthy subjects and HIV-seropositive patients, we described a peculiar pattern and interesting variations in the values of NAD, IMP, GHP, ADP, GDP, ATP, GTP C1-41. In order to establish the metabolic reasons for these changes, we evaluated nucleosides and nucleobasea aa a measure of catabolic rate. A simple method, based on fast ion-pair reversed-phase HPU: was taken from the literature C51, it permits complete and simultaneous separation of purine nucleotides, nucleoaides and nucleobases in only 20 min. Here we report the preliminary data on nucleoside and nucleobase content in human peripheral (PBL) deduced from these blood lymphocytes determinations. For the preparation of lymphocytes, blood was taken at 8 am after overnight fasting. 10 ml of heparinized blood from healthy subjects was usually sufficient for the determination. The lymphocytes were prepared as already reported [l-31, and the pellet was resuspended with ice-cold 0 . 4 N perchloric acid C1-31. For the chromatographic determinations we used a Beckman System Gold Apparatus, equipped with a variable wevelength detector, set at 254 nm, and a prepacked 3 um Beckman C18 column ( 7 . 5 cm x 4.6 m i.d.). The chromatographic conditions are reported in
Table 1. Table 1. Chromatographic conditions. BUFFERS: (A) Low-ionic strength
(B) Low-ionic strength
GRAD1ENT :
FIMJ RATE:
100 mM potassium dihydrogen phosphate pH 6.0 8 mM tetrabutylanrmonium hydrogen sulphate 100 mM potassium dihydrogen phosphate pH 6.0 8 mM tetrabutylammonium hydrogen sulphate 30 X (v/v) methanol 2.5 2.5 5.0 3.0
min min min min 5.0 min 4 . 0 min
A B B B 100 X B down to 0 % B 100 X up to 20 % up to 40 X up to 100 X
1.5 ml/min
The analyses were performed at room temperature. 1 . 5 x 10'3 cells was injected, overloading the injection loop with 50 u l . Peak identities were confirmed by coelution with internal standards and detection at different wavelengths. The pattern of a standard mixture is reported in Fig.1. In addition to purine nucleotides, nucleosides and nucleobases, the oxidized and reduced pyridine coenzymes were also separated, but, obviously, in our FCA extract NADPH and NADH were not detectable since they are easily oxidized. The xanthine peak was also absent because lymphocytes do not exhibit xanthine 20 u1 of extract, corresponding to
0
I
0
"
m
minuks
Fig.1. Ion-pair reversed-phase HPU: of a standard mixture using the chromatographic conditions reported in Table 1. Table 2. Levels of W i n e nucleotides, nucleoaides and nucleobaaes in PBL from healthy subjects and AIDS patientb. Values, given as p~ol/lO'j cells, are the mean + S.E. of 4 determinations. METABOLITE: SUBJECE
A Hypoxanthine Inosine aUanosine
IMP NAD Adenosine
AMP GDP
NADP ADP GTP ATP
115 .L 85 i 21k 85 t 75 k 19* 63 f 88 f 3 t 587 f 220 i 990 i
B 41 21
8 17 15 7 25 22
1 126
30 93
C
95 f 13 90 f 17 49 f 11 41 f 6 1 2 ~ 5 14f 2 48 f 10 92 f 21 32 i 6 36 k 8 1 6 f 5 20;t 3 30 f 18 46 f 21 49 f 15 179 L 38 7;t 3 20* 7 74 i 5 579 f 160 109 f 43 355 ;t 70 776 i 78 1431 f 137
SUBJECTS: A-healthy; 8-asymptomatic;
C-symptomatic
The purine nucleotide, nucleoside and nucleobaae content of peripheral blood lymphocytes from healthy subjects and AIDS patients is reported in Table 2. The HPU: procedure adopted allows the simultaneous evaluation of most purine compounds, giving more and better information than other methods tested to date, which require comparative analysis. The results obtained in AIDS subjects confirm the already reported data [l-41 on nucleotide levels, except for NAD IMP and ADP. Nucleosidea content did not vary significantly in sick patients (except inosine which decreased), neither did hypxanthine levels change. Consequently the generally increased nucleotide content occurring during the infection, might be related to accelerated synthesis. This work was financed by the Italian Secretariat for Health, National Institute of Health, IV AIDS Project, Rome, Italy. References I. Tabucchi, A., Carlucci, F., Ramazzotti. E., Re, M.C., Marinello, E., Rubino, M., Pagani, R. (1991) Biomed. & Pharmacother. 45, 25-29. 2 . Carlucci, F., Tabucchi, A., Consolmagno, E., Tagliaferri, P., Porcelli, B., Marinello, E.. Leoncini, R., Pagani, R. (1992) Biomed. & Phamnacother. 46, 7-12. 3. Tabucchi, A., Carlucci, F., Rubino. M., Marinello, E., Ramazzotti, E., Re. M.C., Pagani, R., (1991) Giorn.It.Chim.Clin. 16, 231-237. 4. Tabucchi, A., Carlucci, F., Marinello, E., Re, M.C., Bisozzi, L., Cacopardo, F., Pagani, R. (1991) Rec.Progr.Med. 82, 581-584. 5. Stocchi, V., Cucchiarini, L., Canestrari, P., Piacentini, M.P., Fornaini, G . (1987) Anal.Biochem. 167. 181-190.
