Biomed % Pharmacother(1992) 46.2529 0 Elsevier, Paris
ossi
Analysis of purine nucleotides in lymphocytes from healthy subjects and A A Tabucchi’,
F Carlucci’, E Ramazzotti2, MC Re2, F Marinello’*, M Rubino3, R Pagani’
‘Istituto di Biochimica e di Enrimologia. Universitk di Siena. Pian dei Mantellini. 44. 53100 Siena; ‘lstituto di Microbiologia, Universitridi Bologna: ‘Divisione Malattie Infettive, Ospedale S Maria della Scala, Siena. Italy (Received 6 January 1992; accepted 17 January 1992)
Summary - The moat important purine nucleotides (NAD, AMP. IMP, GMP, XMP, ADP, ATP, GDP. GTP) were anaIy by HPLC in the lymphocytes of healthy subjects and HIV-1 seropositive patients at different stages of the disease (Al AIDS). Several differences, which focus attention on the behaviour of purine nucleotide metabolism in the Iymphoc: of these patients, were observed. purine nucleotides ! lymphocytes
/ AIDS
RCumi - Analyse des nucldtitidespuriniquesdans les lymphocytesde sujets saints et de maladesatteints de SIDA. principaux nuclkotides puriniques (NAD, AMP, IMP GMP, XMP? ADP, ATP, GDP, GTP) ont &5 analysis par HPLC d les lymphocytes de sujets normaux et chez des s&opositifs VIHI, ri dgfkents stades de l’infecrion. On a observe plusit difft+ences qui at:irent l’attention sur 1’Pvolution du m&tabolisme &s nuclt!otides puriniques &nr les lymphocytes de patients. nuclkotides puriniques I lymphocytes
I SIDA
Introduction * Correspondence and reprints Abbreviations :A. adenine nucleotides; ADP, adenosine 5’-diphosphate: AIDS, acquired immunodeficiency syndrome; AMP, adenosine 5’-monophosphate; ARC, AIDS-related complex; ATP. adenosine 5’-triphosphate; dADP. deoxyadenosine 5’-diphosphate; dATP, deoxyadenosine 5’-triphosphate; dGDP, deoxyguanosine %’ -diphosphate; dGTP, deoxyguanosine 5’triphosphate; EC, energy charge; G, guanine nucleotides; GDP. guanine 5’-diphosphate; GMP, guanosine 5’-monophosphate: GTP, guanosine S-triphosphate: HPLC, high pressure liquid chromatography; IMP, inosine 5’-monophosphate; LSM, lymphocyte separation medium; NAD, nicotinamide adenine dinucleotide; PBL. peripheral blood lymphocytes: PBS, phosphate buffered saline; SCID, severe congenital immunodeficiency; XMP. xanthosine 5’-monophosphate.
Purine metabolism and the purine nucleotide p may vary in AIDS patients in response to the m tiple factors which cause depletion of lymp cytes. The large quantity of blood necessary studying these cells and their low count in AI patients have restricted research in this field. Some preliminary indications may be found the literature. An RNA-dependent virus is volved in the disease and the accumulation large amounts of unintegrated viral DNA has bc observed in infected cells [6]. Altered levels
26
cyclic nuclectides [13] and clear changes in the activities of two enzymes S-N and ADA in the same conditions [lo- 121 have also been reported, indicating that alterations in purine nucleotide metabolism really occur in HIV-I infected lymphocytes and that the problem deserves extensive investigation. One of the parameters for studying purine nucleotide metabolism is the evaluation of purine nucleotide intracellular content. We adopted a known procedure [3, 7-9, 14, 151 for the analysis of this parameter in healthy subjects; we have standardized the normal reference values, focusing attention on a certain degree of variability; all data have been described in another paper [2]. We now report the first determinations in asymptomatic subjects and ARC/AIDS patients.
aterials and methods The nucleotides employed as standards were from Sigma Company Chemical Corporation (St Louis, MI, USA). LSM and PBS were purchased from the Flow Company (Ayrshire, UK). All other chemical reagents were of analytical grade from Merck (Darmstadt. Germany). HPLC solvents were filtered through 0.4%pm membrane filters (Sartorius, GmbH, Giittingen, Germany) before use.
Healthy subjects and patients The subjects were 15 healthy controls - 9 males and 6 females - aged from 26 to 52 years, on normal diet and exercise, 5 asymptomatic HIV-l seropositive subjects, 2 ARC and 7 AIDS patients. The ARC and AIDS patieurj were 9 males and 5 females, aged from 25 to 52 years, grouped according to Walter Reed classification (WRl: 5 patients; WR5: 2 and WR6: 7). Blood samp!cs were analyzed for hemoglobin (which was always normal), white blood cells (2 000-10 000) and platelets (normal in all cases). The CD4+/CD8’ ratio was between 0.46 and 1.00 in asymptomatic subjects: between 0.45 and 0.20 in ARC patients and between 0.01 and 0.46 in AIDS patients. None of the patients had Kaposi’s sarcoma and had not taken drugs for a month.
Preparation
of lymphocytes
The lymphocytes were isolated from heparinized blood taken at 8 am after overnight fasting, using Bdyum’s procedure 111. The pellet was suspended in PBS (40 x IO6 cells/ml). The lymphocytes were counted in a cell counter (Delcon Instruments). No contamination by platelets or erythrocytes was evident. The indications of the Korte
er al [4, 51 were followed for CF!‘Ipreparation, and viability testing.
handling
Nucleotide analysis The lymphocytes were sedimented by centrifugation. the supematant removed and the pellet resuspended in ice- co!d 0.4 N perchloric acid (200 pl for 10 x lo6 cells). The extract was left at O°C for 15 min. mixed and subsequently centrifuged for 10 min at 8 000 g at 4°C. The acid-soluble fraction was brought to pH 6.8 with K$ZOs. Potassium perchlorate was eliminated by centrifugation. The nucleotides were analyzed by high pressure liquid chromatography (HPLC). We used a Beckman Sys-
tem Gold Chromatograph, equipped with two Mod1 IOB pumps, a variable wavelength detector and a prepacked Partisil 10 SAX stainless steel column (250 x 46 mm id). One hundred pl of extract, corresponding to - 5 x 10” cells was injected, overloading the injection loop with 130 pI. All the elution conditions, the resolution of the most important nucleotides (NAD, AMP, IMP, GMP, XMP, ADP, GDP, ATP, and GTP) and their determination in duplicate were as described by De Korte et al [4, 51. The results were in good agreement with the data of these authors. Nucleotide detection was carried out at 254 nm, to reduce a specific interference. We only investigated the above reported compounds, ignoring pyrimidine nucleotides and other peaks which were either too low or not constant, or beyond the scope of this study. Statistical analysis of the data was performed by oneway analysis of variance, taking P 5 0.05 as the significant level.
Results Typical chromatograms from a healthy subject and an AIDS patient are reported in figures 1 and 2. Table I shows nucleotide levels in healthy subjects and the different categories of patients. The normal levels found through the present research confirm both the already reported reference values [2] and the high SE: despite the observed variability, some differences were clearly significant and we have taken them into specific consideration. In asymptomatic HIV-l seropositive subjects, NAD was unchanged and the concentration of IMP was enhanced; none of the other monophosphates showed significant variations. Ribonucleoside di- and tri-phosphates ADP, GDP, ATP and GTP, were significantly depressed. In ARC patients (who were too few to permit definite conclusionsl. the oattern of mononu-
27
minutes
ig 1. Typical HPLC nucleotide
profile of a lymphocyte exact from a healthy subject. i;or nucleotide extraction see
‘aterials and methods.
Fig 2. Typical HPLC nucleotide profile of a lymphocyte extract from an AIDS patients.
‘able I. Puke nucleotide content in peripheral blood lymphocytes. Subjects
Iealthy
No of cases
15
kymptomatic
5
9RC 9IDS
2 7
NAD
264 f80 276 f 68 142 469* AZ121
AMP
23 +6 31 + 10 22 21 6
IMP
GMP
XMP
28 f8 96* +15 55 52. f10
10 f3 18 f6 23 11 It5
6 +2 0.5 +0 0.5 14 +6
ADP
GDP
409 -+67 209* 540 408 681* + 180
57 +9 35* f6 89 115* + 30
ATP
GTP -._
686 flG7 540* + 62 1288 1466* f 250
173 +9 132” + 1’5 348 336* + 77
.-.
‘alues are mean + SE in pmol/106 cells; for experimental conditions,see Materialsand merhmis;* P e 0.05 with respectto normal subjects
leotides seemed to be unchanged with respect to ealthy subjects; NAD was lower, ADP and GDP rere close to normal values and ATP and GTP lere higher than in seropositive and normal lymhocytes. In the lymphocytes of AIDS patients, NAD was ignificantly higher, monophosphates were not ignificantly different, and di- and k-phosphates lere significantly higher than in healthy subjects.
E.C.AMP
E.C.GMP
I
1
ATP/AOP
No correlation was found between nucleotide :vels and CD4+/CDS+ ratio at the various stages f the disease. Figure 3 shows some parameters which are onsidered from the literature [3-5, 7, 141 as an ldex of the energy and viability of the cells. The energy charge for adenylate and guanylate about 0.80 - was substantially consistent with
Fig 3. Some important parameters and ratios between purine nucleotides in PBLfrom healthy subjects and patients at different stages of HIV-I infection.
28 that found by other authors [3-5, 7, 141. The ATP/ADP ratio was about 2, as is the case when lymphocytes are prepared from heparinized blood 151. It is very interesting to note that all such parameters were substantially unchanged in the lymphocytes of all patients; a tendency of the ATP/ADP, GTP/GDP ratios to increase in the disease was observed.
iscussion In interpreting our results, it is necessary to distinguish the reasons for the variations observed and their mechanism. The reasons are multiple; apart from the penetration of an RNA-virus in a cell, even if it appears to be very poorly expressed at least in PBL [6], it should be remembered that the T-cells I’rom AIDS patients are characterized by defective activation [ 131 and ‘that the humoral relationships (interleukin-2 and others) between lymphocytes are modified by HIV-l [6]. All these factors could act either on the genomic or enzymatic levels and are related to genera! i;r~t!;rr.~ elf the disease, beyond the purpose of the present research. In order to understand the mechanism, it should be remembered that an increase in metabolites can be ascribed either to an accelerated synthesis or to minor degradation and utilization. The increase in IMP might be linked to the decrease of 5’ -N and to the increase in ADA observed in such conditions [IO-121; decrease in diand tri-phosphates. in seropositive subjects, can be due to accelerated degradation and utilization; this could explain the accumulation of unintegrated DNA [6], the increase in cyclic nucleotides 1131 and the formation of viral RNA [6] in infected lymphocytes. On the other hand, the increase in NAD, ADP, ATP, GDP, GTP in AIDS patients could be interpreted on the basis of an accelerated synthesis, and be related to the impaired activation of the lymphocytes 1131. These hypotheses must be verified by following both the kinetics of the into oration of labelled precursors (‘4C-formate and ‘p4C-glycine) into purine nucleotides, and the activity of the enzymes involved both in the de IZOVOsynthesis and in the salvage pathway. Independently of a final interpretation following further experimentation, our study suggests
that msny biochemical problems regarding virusinfected lymphocytes deserve investigation. If we rem*:mbe- the contributions made by research [ 161 on purine nucleotide metabolism, towards correct diagnosis and treatment of other diseases of the lymphocytes (SCID, leukemia, mononucleosis), we can conclude that further investigation in these fields, including AIDS, might be a useful perspective opened up by the present study.
Acknowledgment This work was supported by the Italian Secretariar for Health, National Institute 1990.
of Health, 3rd AIDS project,
eferences Biiyum A (1968 Isolation of leukocytes from human blood. Further observations. Stand J Ciin Lab Invest 21 suppl 97, 31 Carlucci F, Tabucchi A. Consolmagno E, Porcelli B, Marine110 E, Leoncini R, Pagani R (1992) Level and variability of purine nucleoti&s in normal human lymphocyte. Biomed & Pharrlacother 46, in press De Abreu RA, Pethers GJ, Veerkampf GH (1982) Concentration of nucleotides in peripheral blood lymphocytes of various mammalian species. Adv Esp Med Biol 165B, 125 De Korte D, Haverkort WA, van Gennip AH, Roos D (1985) Nucleotide profiles of normal human blood cells determined by high performance liquid chromatography. Anal Biocirem 147, 197 De Korte D, Haverkort WA, Roos D, van Gennip AH (1985) Anion excha:ige high performance liquid chromatography method for the quantitation of nucleotides in human blood cells. Clin Chim Acra 148, 185 Fauci AS (1988) The human immunodeficiency virus: infectivity and mechanism of pathogenesis. Science 239, 617 Goday A, Simmonds HA, Webster DR, Levinsky RJ, Watson AR, Hoffbrand AF (1983) Importance of platelet - free preparations for evaluating lymphocyte nucleotide levels in inherited or acquired immunoldeficiency syndromes. Clin Sci 65, 635 Marijnen YMT, de Korte D, Haverkort WA, den Breejen EJS, van Gennip AH, Roos D (1989) Studies on the incorporation of precursors into purine and pyrimidine nucleotides via de novo and “salvage” pathways in normal lymphocytes and lymphoblastic cell lines. Biocltim Siophys Acta 1012. 148
29 Marinello E. Pagani R. Carlucci F, Molinelli M. Valerio P, Tabucchi A (1991) The purine nucleotide content in normal human lymphocytes. Biochenz Sot Tram 19, 3478 Murray JL, Reuben JM, Munn CG, Manscll PWA, Newell GR, Hersh EM (1984) Decreased 5’ -nucleotidase activity in lymphocytes from asymptomatic sexually active homosexual men and patients with acquired deficiency syndrome. Blood 64, 1016 Murray JL, Loftin KC, Munn CC, Reuben MR. Manse11 PW. Hcrch EM (1985) Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocyte from patients with the acquired immunodcficicncy syndrome. Blood 6, 13 18 Murray JL. Bywaters DW, Reuben JM. Mansell PW, Hersh EM (1987) Decreased 5’ -nuclcotidasc activity in suppressor (OKT8) T-lymphocytes from homosexuals with AIDS related complex: Nonasso-
ciation with enhanced deoxynucleoside Clin Immunol Immunopatlrol 42, 10
toxicity.
13 Nokta M, Pollard R (1991) Human immunodeficicncy virus infection: association with altered intracellular levels of CAMP and cGMP in MT-4 cells. Virology 181, 211 14 Peters GJ, De Abreu RA. Oosterhof A, Veerkampf JH (1983) Concentration of nucleotidcs and deoxynucleotides in peripheral and phytohemagglutinin stimulated mammalian lymphocytes. Biochim Biophys Acia 759, 7 15 Spaapen LJM, Scharenberg JGM, Zegers BJM, Riskers GT, Duran M, Wadman SK (1985) Intracellular purine and pyrimidine nucieotide pools of human T and B lymphocytes. Adv Exp Med Biof 195A. 567 16 Watts RWV (1987) Purine enzymes function. Clin Bioclrcm Rev 239
and immune
ossi
Analysis of purine nucleotides in lymphocytes from healthy subjects and A A Tabucchi’,
F Carlucci’, E Ramazzotti2, MC Re2, F Marinello’*, M Rubino3, R Pagani’
‘Istituto di Biochimica e di Enrimologia. Universitk di Siena. Pian dei Mantellini. 44. 53100 Siena; ‘lstituto di Microbiologia, Universitridi Bologna: ‘Divisione Malattie Infettive, Ospedale S Maria della Scala, Siena. Italy (Received 6 January 1992; accepted 17 January 1992)
Summary - The moat important purine nucleotides (NAD, AMP. IMP, GMP, XMP, ADP, ATP, GDP. GTP) were anaIy by HPLC in the lymphocytes of healthy subjects and HIV-1 seropositive patients at different stages of the disease (Al AIDS). Several differences, which focus attention on the behaviour of purine nucleotide metabolism in the Iymphoc: of these patients, were observed. purine nucleotides ! lymphocytes
/ AIDS
RCumi - Analyse des nucldtitidespuriniquesdans les lymphocytesde sujets saints et de maladesatteints de SIDA. principaux nuclkotides puriniques (NAD, AMP, IMP GMP, XMP? ADP, ATP, GDP, GTP) ont &5 analysis par HPLC d les lymphocytes de sujets normaux et chez des s&opositifs VIHI, ri dgfkents stades de l’infecrion. On a observe plusit difft+ences qui at:irent l’attention sur 1’Pvolution du m&tabolisme &s nuclt!otides puriniques &nr les lymphocytes de patients. nuclkotides puriniques I lymphocytes
I SIDA
Introduction * Correspondence and reprints Abbreviations :A. adenine nucleotides; ADP, adenosine 5’-diphosphate: AIDS, acquired immunodeficiency syndrome; AMP, adenosine 5’-monophosphate; ARC, AIDS-related complex; ATP. adenosine 5’-triphosphate; dADP. deoxyadenosine 5’-diphosphate; dATP, deoxyadenosine 5’-triphosphate; dGDP, deoxyguanosine %’ -diphosphate; dGTP, deoxyguanosine 5’triphosphate; EC, energy charge; G, guanine nucleotides; GDP. guanine 5’-diphosphate; GMP, guanosine 5’-monophosphate: GTP, guanosine S-triphosphate: HPLC, high pressure liquid chromatography; IMP, inosine 5’-monophosphate; LSM, lymphocyte separation medium; NAD, nicotinamide adenine dinucleotide; PBL. peripheral blood lymphocytes: PBS, phosphate buffered saline; SCID, severe congenital immunodeficiency; XMP. xanthosine 5’-monophosphate.
Purine metabolism and the purine nucleotide p may vary in AIDS patients in response to the m tiple factors which cause depletion of lymp cytes. The large quantity of blood necessary studying these cells and their low count in AI patients have restricted research in this field. Some preliminary indications may be found the literature. An RNA-dependent virus is volved in the disease and the accumulation large amounts of unintegrated viral DNA has bc observed in infected cells [6]. Altered levels
26
cyclic nuclectides [13] and clear changes in the activities of two enzymes S-N and ADA in the same conditions [lo- 121 have also been reported, indicating that alterations in purine nucleotide metabolism really occur in HIV-I infected lymphocytes and that the problem deserves extensive investigation. One of the parameters for studying purine nucleotide metabolism is the evaluation of purine nucleotide intracellular content. We adopted a known procedure [3, 7-9, 14, 151 for the analysis of this parameter in healthy subjects; we have standardized the normal reference values, focusing attention on a certain degree of variability; all data have been described in another paper [2]. We now report the first determinations in asymptomatic subjects and ARC/AIDS patients.
aterials and methods The nucleotides employed as standards were from Sigma Company Chemical Corporation (St Louis, MI, USA). LSM and PBS were purchased from the Flow Company (Ayrshire, UK). All other chemical reagents were of analytical grade from Merck (Darmstadt. Germany). HPLC solvents were filtered through 0.4%pm membrane filters (Sartorius, GmbH, Giittingen, Germany) before use.
Healthy subjects and patients The subjects were 15 healthy controls - 9 males and 6 females - aged from 26 to 52 years, on normal diet and exercise, 5 asymptomatic HIV-l seropositive subjects, 2 ARC and 7 AIDS patients. The ARC and AIDS patieurj were 9 males and 5 females, aged from 25 to 52 years, grouped according to Walter Reed classification (WRl: 5 patients; WR5: 2 and WR6: 7). Blood samp!cs were analyzed for hemoglobin (which was always normal), white blood cells (2 000-10 000) and platelets (normal in all cases). The CD4+/CD8’ ratio was between 0.46 and 1.00 in asymptomatic subjects: between 0.45 and 0.20 in ARC patients and between 0.01 and 0.46 in AIDS patients. None of the patients had Kaposi’s sarcoma and had not taken drugs for a month.
Preparation
of lymphocytes
The lymphocytes were isolated from heparinized blood taken at 8 am after overnight fasting, using Bdyum’s procedure 111. The pellet was suspended in PBS (40 x IO6 cells/ml). The lymphocytes were counted in a cell counter (Delcon Instruments). No contamination by platelets or erythrocytes was evident. The indications of the Korte
er al [4, 51 were followed for CF!‘Ipreparation, and viability testing.
handling
Nucleotide analysis The lymphocytes were sedimented by centrifugation. the supematant removed and the pellet resuspended in ice- co!d 0.4 N perchloric acid (200 pl for 10 x lo6 cells). The extract was left at O°C for 15 min. mixed and subsequently centrifuged for 10 min at 8 000 g at 4°C. The acid-soluble fraction was brought to pH 6.8 with K$ZOs. Potassium perchlorate was eliminated by centrifugation. The nucleotides were analyzed by high pressure liquid chromatography (HPLC). We used a Beckman Sys-
tem Gold Chromatograph, equipped with two Mod1 IOB pumps, a variable wavelength detector and a prepacked Partisil 10 SAX stainless steel column (250 x 46 mm id). One hundred pl of extract, corresponding to - 5 x 10” cells was injected, overloading the injection loop with 130 pI. All the elution conditions, the resolution of the most important nucleotides (NAD, AMP, IMP, GMP, XMP, ADP, GDP, ATP, and GTP) and their determination in duplicate were as described by De Korte et al [4, 51. The results were in good agreement with the data of these authors. Nucleotide detection was carried out at 254 nm, to reduce a specific interference. We only investigated the above reported compounds, ignoring pyrimidine nucleotides and other peaks which were either too low or not constant, or beyond the scope of this study. Statistical analysis of the data was performed by oneway analysis of variance, taking P 5 0.05 as the significant level.
Results Typical chromatograms from a healthy subject and an AIDS patient are reported in figures 1 and 2. Table I shows nucleotide levels in healthy subjects and the different categories of patients. The normal levels found through the present research confirm both the already reported reference values [2] and the high SE: despite the observed variability, some differences were clearly significant and we have taken them into specific consideration. In asymptomatic HIV-l seropositive subjects, NAD was unchanged and the concentration of IMP was enhanced; none of the other monophosphates showed significant variations. Ribonucleoside di- and tri-phosphates ADP, GDP, ATP and GTP, were significantly depressed. In ARC patients (who were too few to permit definite conclusionsl. the oattern of mononu-
27
minutes
ig 1. Typical HPLC nucleotide
profile of a lymphocyte exact from a healthy subject. i;or nucleotide extraction see
‘aterials and methods.
Fig 2. Typical HPLC nucleotide profile of a lymphocyte extract from an AIDS patients.
‘able I. Puke nucleotide content in peripheral blood lymphocytes. Subjects
Iealthy
No of cases
15
kymptomatic
5
9RC 9IDS
2 7
NAD
264 f80 276 f 68 142 469* AZ121
AMP
23 +6 31 + 10 22 21 6
IMP
GMP
XMP
28 f8 96* +15 55 52. f10
10 f3 18 f6 23 11 It5
6 +2 0.5 +0 0.5 14 +6
ADP
GDP
409 -+67 209* 540 408 681* + 180
57 +9 35* f6 89 115* + 30
ATP
GTP -._
686 flG7 540* + 62 1288 1466* f 250
173 +9 132” + 1’5 348 336* + 77
.-.
‘alues are mean + SE in pmol/106 cells; for experimental conditions,see Materialsand merhmis;* P e 0.05 with respectto normal subjects
leotides seemed to be unchanged with respect to ealthy subjects; NAD was lower, ADP and GDP rere close to normal values and ATP and GTP lere higher than in seropositive and normal lymhocytes. In the lymphocytes of AIDS patients, NAD was ignificantly higher, monophosphates were not ignificantly different, and di- and k-phosphates lere significantly higher than in healthy subjects.
E.C.AMP
E.C.GMP
I
1
ATP/AOP
No correlation was found between nucleotide :vels and CD4+/CDS+ ratio at the various stages f the disease. Figure 3 shows some parameters which are onsidered from the literature [3-5, 7, 141 as an ldex of the energy and viability of the cells. The energy charge for adenylate and guanylate about 0.80 - was substantially consistent with
Fig 3. Some important parameters and ratios between purine nucleotides in PBLfrom healthy subjects and patients at different stages of HIV-I infection.
28 that found by other authors [3-5, 7, 141. The ATP/ADP ratio was about 2, as is the case when lymphocytes are prepared from heparinized blood 151. It is very interesting to note that all such parameters were substantially unchanged in the lymphocytes of all patients; a tendency of the ATP/ADP, GTP/GDP ratios to increase in the disease was observed.
iscussion In interpreting our results, it is necessary to distinguish the reasons for the variations observed and their mechanism. The reasons are multiple; apart from the penetration of an RNA-virus in a cell, even if it appears to be very poorly expressed at least in PBL [6], it should be remembered that the T-cells I’rom AIDS patients are characterized by defective activation [ 131 and ‘that the humoral relationships (interleukin-2 and others) between lymphocytes are modified by HIV-l [6]. All these factors could act either on the genomic or enzymatic levels and are related to genera! i;r~t!;rr.~ elf the disease, beyond the purpose of the present research. In order to understand the mechanism, it should be remembered that an increase in metabolites can be ascribed either to an accelerated synthesis or to minor degradation and utilization. The increase in IMP might be linked to the decrease of 5’ -N and to the increase in ADA observed in such conditions [IO-121; decrease in diand tri-phosphates. in seropositive subjects, can be due to accelerated degradation and utilization; this could explain the accumulation of unintegrated DNA [6], the increase in cyclic nucleotides 1131 and the formation of viral RNA [6] in infected lymphocytes. On the other hand, the increase in NAD, ADP, ATP, GDP, GTP in AIDS patients could be interpreted on the basis of an accelerated synthesis, and be related to the impaired activation of the lymphocytes 1131. These hypotheses must be verified by following both the kinetics of the into oration of labelled precursors (‘4C-formate and ‘p4C-glycine) into purine nucleotides, and the activity of the enzymes involved both in the de IZOVOsynthesis and in the salvage pathway. Independently of a final interpretation following further experimentation, our study suggests
that msny biochemical problems regarding virusinfected lymphocytes deserve investigation. If we rem*:mbe- the contributions made by research [ 161 on purine nucleotide metabolism, towards correct diagnosis and treatment of other diseases of the lymphocytes (SCID, leukemia, mononucleosis), we can conclude that further investigation in these fields, including AIDS, might be a useful perspective opened up by the present study.
Acknowledgment This work was supported by the Italian Secretariar for Health, National Institute 1990.
of Health, 3rd AIDS project,
eferences Biiyum A (1968 Isolation of leukocytes from human blood. Further observations. Stand J Ciin Lab Invest 21 suppl 97, 31 Carlucci F, Tabucchi A. Consolmagno E, Porcelli B, Marine110 E, Leoncini R, Pagani R (1992) Level and variability of purine nucleoti&s in normal human lymphocyte. Biomed & Pharrlacother 46, in press De Abreu RA, Pethers GJ, Veerkampf GH (1982) Concentration of nucleotides in peripheral blood lymphocytes of various mammalian species. Adv Esp Med Biol 165B, 125 De Korte D, Haverkort WA, van Gennip AH, Roos D (1985) Nucleotide profiles of normal human blood cells determined by high performance liquid chromatography. Anal Biocirem 147, 197 De Korte D, Haverkort WA, Roos D, van Gennip AH (1985) Anion excha:ige high performance liquid chromatography method for the quantitation of nucleotides in human blood cells. Clin Chim Acra 148, 185 Fauci AS (1988) The human immunodeficiency virus: infectivity and mechanism of pathogenesis. Science 239, 617 Goday A, Simmonds HA, Webster DR, Levinsky RJ, Watson AR, Hoffbrand AF (1983) Importance of platelet - free preparations for evaluating lymphocyte nucleotide levels in inherited or acquired immunoldeficiency syndromes. Clin Sci 65, 635 Marijnen YMT, de Korte D, Haverkort WA, den Breejen EJS, van Gennip AH, Roos D (1989) Studies on the incorporation of precursors into purine and pyrimidine nucleotides via de novo and “salvage” pathways in normal lymphocytes and lymphoblastic cell lines. Biocltim Siophys Acta 1012. 148
29 Marinello E. Pagani R. Carlucci F, Molinelli M. Valerio P, Tabucchi A (1991) The purine nucleotide content in normal human lymphocytes. Biochenz Sot Tram 19, 3478 Murray JL, Reuben JM, Munn CG, Manscll PWA, Newell GR, Hersh EM (1984) Decreased 5’ -nucleotidase activity in lymphocytes from asymptomatic sexually active homosexual men and patients with acquired deficiency syndrome. Blood 64, 1016 Murray JL, Loftin KC, Munn CC, Reuben MR. Manse11 PW. Hcrch EM (1985) Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocyte from patients with the acquired immunodcficicncy syndrome. Blood 6, 13 18 Murray JL. Bywaters DW, Reuben JM. Mansell PW, Hersh EM (1987) Decreased 5’ -nuclcotidasc activity in suppressor (OKT8) T-lymphocytes from homosexuals with AIDS related complex: Nonasso-
ciation with enhanced deoxynucleoside Clin Immunol Immunopatlrol 42, 10
toxicity.
13 Nokta M, Pollard R (1991) Human immunodeficicncy virus infection: association with altered intracellular levels of CAMP and cGMP in MT-4 cells. Virology 181, 211 14 Peters GJ, De Abreu RA. Oosterhof A, Veerkampf JH (1983) Concentration of nucleotidcs and deoxynucleotides in peripheral and phytohemagglutinin stimulated mammalian lymphocytes. Biochim Biophys Acia 759, 7 15 Spaapen LJM, Scharenberg JGM, Zegers BJM, Riskers GT, Duran M, Wadman SK (1985) Intracellular purine and pyrimidine nucieotide pools of human T and B lymphocytes. Adv Exp Med Biof 195A. 567 16 Watts RWV (1987) Purine enzymes function. Clin Bioclrcm Rev 239
and immune
