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The levels of adenine nucleotides and pyridine coenzymes in red blood cells from the newborn, determined simultaneously by HPLC
131
Clinica Chimica Acta, 189 (1990) 131-138 Elsevier
CCA 04735
The levels of adenine nucleotides and pyridine coenzymes in red blood cells from the newborn, determined simultaneously by HPLC Marilena Formato’,
Bruno Masala 1 and Giancarlo De Luca *
’ Institute of General Physiology and Biological Chemistry and 2 Institute of Applied Biology, University of Sassari, Sassari (Italy) (Received
11 February
1989; revision received
Key words: Newborn
RBC; Adenine
12 July 1989; accepted
nucleotide;
Pyridine
13 March
coenzyme;
1990)
HPLC
The concentrations of ATP, ADP, AMP; NADP and NADPH; NAD and NADH were determined in erythrocytes from healthy newborns and compared with those obtained in healthy adults. No significant differences were found for the adenine nucleotide concentrations, but NADH levels were reduced in newborn erythrocytes, with a consequent increase in the NAD/NADH ratio. Moreover, in newborn erythrocytes increased levels of NADP were observed, with a consequent increase in the NADP/NADPH ratio and a decrease in the NAD/NADP ratio. These results indicate the need to use reference values of the ratios NAD/NADH, NADP/NADPH and NAD/NADP from healthy newborns in the study of syndromes affecting the metabolism of erythrocytes in the newborn.
Introduction The determination of the levels of adenine nucleotides (ATP, ADP) (AMP) and those of pyridine nucleotides, both in the oxidized and in the reduced form (NAD, NADH; NADP, NADPH), gives important information on the energetic and redox metabolism of the cell. For instance, in normal human erythrocytes (RBC) the measurement of the energy charge is an index of cell integrity [l]. The adenine nucleotide pool is modified in RBC of subjects affected with different pathological conditions [2-61, and a decrease of ATP concentration was observed during cell ageing [7-131. The measurement of pyridine nucleotides and the determination of
Correspondence to: Prof. Bruno MasaIa, Istituto di Fisiologia di Sassari, Via Muroni 25, I-07100 Sassari, Italy.
0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
Generale
e Chin&a
B.V. (Biomedical
Division)
Biologica,
Universita
132
NAD/NADH and NADP/NADPH ratios indicate the efficiency of glycolysis and the pentose-phosphate shunt. Moreover, the NADP/NADPH ratio has been found to be significantly altered in RBC of patients affected with hemolytic anemias [14,15]. It is still not known whether these parameters vary with the age of the subject, although this information may be critical in the study of any pathological condition affecting the newborn. For this reason we determined the concentrations of adenine nucleotides and pyridine coenzymes in RBC from normal human newborns by means of a recently developed reversed-phase HPLC method, which allows their simult~eous dete~nation after alkaline extraction [16,17]. Materials and methods The blood samples were obtained by venepuncture from 16 healthy newborns (within 2 days of delivery), and from 13 healthy adults (with a mean age of 30 yr) and collected in EDTA as ~tico~~~t. Ethical approval for the blood samples was obtained from both parents of each of the newborns. A micromethod employing 75 x 1 mm i.d. capillary tubes was used for hematocrit estimation. Samples were subjected to alkaline extraction of nucleotides within approximately 1 h. Alkaline extraction of nucleotides from whole blood was carried out according to Stocchi et al. [16,17]: 1 ml of blood was added to 1 ml of ice%old 0.5 mol/l KOH to deproteinize and filtered by centrifugation at 2 500 x g x 10 min at 4OC on a CF SOA Amicon membrane (Amicon, Lexington, MA, USA). The clear ultrafiltered solution was adjusted to pH 6.5 with 1 mol/l KH,PO., and immediately used for HPLC analysis. Portions were also stored at - 80” C and analysed after different periods. Reproducible results were obtained in samples stored up to 3 mth. Nucleotides were separated and determined essentially as described by Stocchi et al. [16,17] by means of an HPLC system (LKB, Bromma, Sweden) equipped with a LKB LyChrosorb RP-18 column (250 X 4.6 mm i.d.), protected with a guard column filled with pellicular reverse-phase material. The injection volume of the alkaline-extracted sample was 20 ~1. The eluent consisted of 2 elements: 0.1 mol/l KH,PO,, pH 6.0 (buffer A), and 0.1 mol/l KH,PO, containing 2.5 mol/l CH,OH, pH 6.0 (buffer B). The most suitable gradient was 11 min at 100% buffer A, 8 min from 100% buffer A to 25% buffer B, 8 min from 25% to 90% buffer B, 6 min from 90% to 100% buffer B, and finally 6 min at 100% buffer B. The flow rate was 1 ml/mm at room temperature, and detection was at 254 nm. Identity of the peaks was confirmed by coelution with standards (Sigma, St. Louis, MO, USA). Quantitative dete~nations were performed by injecting standard solutions of known concentration. Concentration of adenine and pyridine nucleotides was expressed as mmol/I RBC, using the hematocrit to correct the whole blood volume. Energy charge (EC) value was determined according to the equation: EC = (ATP + 1/2ADP)/(ATP + ADP + AMP). An analysis of the marginal distribution of individual variables was performed before the calculation of mean, standard deviation and Student’s t test. The degree of conformity to the normal dist~bution was evaluated via beets and kurtosis coefficients. A symmetrizing transformation was applied to the varia-
133
bles showing a significant asymmetry. Student’s t test was used to determine the significance of differences between the mean of the two groups for each of the variables. LOO
0
10
20
30
40
TIME (min)
Fig. 1. Simultaneous reversed-phase HPLC separation of adenine nucleotides and pyridme coenzymes. A, Alkaline extract from human adult RBC; B, alkaline extract from human newborn RBC. Due to the low concentration of some nucleotides in erythrocyte specimens, the sensitivity of the U.V. detector was increased from 0.05 to 0.01 absorbance unit of full scale (A.U.F.S.) after elution of ATP and ADP.
134
Results
Figure 1 shows the separation of adenine nucleotides and pyridine coenzymes in RBC from adults and newborn infants. The concentrations of adenine nucleotides and pyridine coenzymes (together with the values of EC, NAD/NADH, NADP/NADPH and NAD/NADP ratios) in these subjects are shown in Tables I and II, respectively. A few variables (NADH, NAD/NADH, ADP in the case of adults; NADH, NAD/NADH, NADP/NADPH, ADP in the case of newborns) were asymmetric on the original scale, but did not show appreciable asymmetry once transformed on a logarithmic scale. Means and 95% reference limits for these variables were calculated on the transformed scale. The adult RBC concentration of both adenine and pyridine nucleotides, EC value, and the ratios of oxidized/reduced coenzymes were in agreement with those described previously [16,17]. Neither adenine nucleotide concentrations nor EC value obtained in neonatal RBC differ significantly from those of adults. On the contrary, neonatal NADH levels appeared to be reduced and in 6 out of the 16 newborns resulted undetectable, so precise determination was impossible. For this reason, we could not include these values in the relevant analysis, obtaining a mean value which resulted non-significantly different from that of adult RBC. Therefore, the actual mean value has to be certainly lower, with a consequent significant increase in the NAD/NADH ratio. Significantly increased levels of NADP and the NADP/NADPH ratio were observed in neonates. Consequently, the NAD/NADP ratio was lower in neonatal RBC. Discussion
The data obtained in this study shows that both adenine nucleotide concentrations and the energy charge in neonatal RBC do not differ significantly from those of adults. On the contrary, in newborn RBC the concentration of NADH was lower, and NAD/NADH ratio consequently higher. Since a good correlation has been re-
TABLE I Mean and 95% reference limits for the concentration of adenine nucleotides and energy charge value in adult and newborn red blood ceIIs
Adults (n =13) Newborns (n =16) t
ATP
ADP
AMP
E.C.
1530 (1160-l 900) 1516 (1168-l 864) -0.2 b
172.43 a (116.51-255.19) 145.5 a (104.30-203.00) - 2.52 b
16.10 (8.89-23.31) 16.75 (7.40-26.10) 0.41 b
0.940 (0.924-0.956) 0.946 (0.934-0.958) 2.41 b
Values are expressed as mmoI/l BBC. * Variable non nomiaBy distributed and transformed in logatithrns. b p > 0.01 non-significant.
135
I
136
ported between the value of NAD/NADH ratio and RBC ability to utilize glucose [18], our results might suggest a higher rate of the glycolytic pathway in the neonatal RBC. This interpretation is also sustained by the findings that the newborn RBC displays higher percentages of young cells than are found in adults [19,20], and that both anaerobic glycolysis [ll] and hexokinase activity [7,9,13,21-241 decrease during RBC ageing. Moreover, the NADP and NADPH determinations in neonates and adults showed significant differences. NADP concentrations were higher in neonates, whereas NADPH levels appeared to be slightly reduced, with a consequent marked increase in the NADP/NADPH ratio. Since NADPH is necessary for regeneration of reduced glutathione in the glutathione reductase reaction, the increase in NADP/NADPH ratio probably reflects a higher NADPH utilization in newborn red cells related to their increased susceptibility to oxidation [25]. The NAD/NADP ratio was also significantly lower in neonatal RBC. This is consistent with the recent report that the NAD/NADP ratio increases in rabbit RBC with ageing [26], since newborn red cells have higher percentage of young cells [19,20] and therefore a shorter life-span. The reason for the reduced life-span of neonatal RBC is not known. If membrane damage by free radicals is of major importance [25], the increased NADP concentration might promote the pentosephosphate shunt and thus provide some protection by increased reducing capacity. We might conclude that, in the study of erythrocyte metabolism in pathological conditions affecting the newborn, it is particularly important to use normal values of NAD/NADH, NADP/NADPH, and NAD/NADP ratios determined in healthy newborn, since they significantly differ from the corresponding values in adult RBC. Acknowledgements
This work was supported by funds from M.P.I. (40%), Italy. We are greatly indebted to Prof. L. Belardinelli, who kindly helped us in statistical analysis. References 1 Atkinson DE. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 1%8;7:4030-4034. 2 Valentine WN, Paglia DE, Tartagba AP, Gilsanz F. Hereditary hemolytic anemia with increased red ceil adenosine deaminase (45- to ‘IO-fold) and decreased adenosine triphosphate. Science 1977;195:783-785. 3 Ericson A, Niklasson F, de Verdier CH. A systematic study of nucleotide analysis of human erythrocytes using an anionic exchanger and HPLC. Chn Chim Acta 1977;127,47-59. 4 Staal GEJ, Jansen G, Ross D. Pyruvate kinase and the ‘High ATP syndrome’. J Clin Invest 1984;74:231-235. 5 Stocchi V, Magnani M, Cucchiarini L, NoveBi G, Dallapiccola B. Red blood ceil adenine nucleotides abnormalities in Down syndrome. Am J Med Gen 1985;20:131-135. 6 Novelh G, Stocchi V, Giannotti A, Magnani M, DaIlapiccola B. Increased erythrocyte adenosine deaminase activity without haemolytic anaemia. Hum Hered 1986;36:37-40. 7 Brewer GX General red ceII metabolism. In: Surgenor D MacN, ed. The red blood cell, Vol I. New York: Academic Press, 1974;387-433.
137 8 Liehtman MA. Does ATP decrease exponentially during red cell ageing? Nouv Rev Fr Hematol 1975;15:625-632. 9 Fomaini G, Magnani M, Dacha M, Bosst M, Stocchi V. Relationship between glucose phosphorylating activities and erythrocytes age. Mech Ageing Dev 1978;8:249-256. 10 Magnani M, Stocchi V, Dacha M, Canestrari F, Fomaini G. Hexokinase isozymic pattern during red cell ageing. FEBS Lett 1980;120:264-266. 11 Seaman C, Wyss S, Piomelli S. The decline in energetic metabolism with ageing of the erythrocyte and its relationship to cell. Am J Hematol 1980;8:31-42. 12 Stocchi V, Magnani M, Canestrari F, Dacha M, Fomaini G. Multiple forms of human red blood cell hexokinase. Preparation, characterization, and age dependence. J Biol Chem 1982;257:2357-2364. 13 Magnani M, Fazi A, Accorsi A, Stocchi V, Dacha M, Fomaini G. Regulatory properties of human erythrocyte hexokinase during cell ageing. Arch Biochem Biophys 1985;239:352-358. 14 Kirkman I-IN, Gaetani GD, Clemons EH, Mareni C. Red cell NADP and NADPH in glucose-6-phosphate dehydrogenase deficiency. J Clin Invest 1975;55:875-878. 15 Magnani M, Stocchi V, Canestrari F, et al. Redox and energetic state of red blood cells in G6PD deficiency, heterozygous beta-thalassemia and the combination of both. Acta Haematol 1986;75:211214. 16 Stocchi V, Cucchiarini L, Magnani M, Chiarantini L, Palma P, Crescentini G. Simultaneous extraction and reverse-phase high-performance chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal Biochem 1985;146:118-124. 17 Stocchi V, Cucchiarini L, Canestrari F, Piacentini P, Fomaini G. A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells. Anal Biochem 1987;167:181-191. 18 Stocchi V, Cucchiarini L, Magnani M, Fomaini G. Adenine and pyridine nucleotides in the erythrccyte of different mammalian species. Biochem Int 1987;14:1043-1053. 19 Masala B, Manta L, Format0 M, Pi10 G. A study of the switch of fetal hemoglobin in newborn erythrocyte fractionated by density gradient. Hemoglobin 1983;7:567-572. 20 Smith JB, Butzner JD, Wills MR, Sabio H, Savory J. Erythrocyte creature in cord blood. Ann Clin Lab Sci 1983;13:439-443. 21 Turner BM, Fischer RA, Harris H. The age related loss of activity of four enzymes in the human erythrocytes. Clin Chim Acta 1974;50:85-95. 22 Rogers PA, Fisher RA, Harris H. An examination of the age-related patterns of decay of the hexokinase of human red cells. Clin Chim Acta 1975;65:291-298. 23 Chapman RG, Shaumberg L. Glycolysis and glycolytic enzyme activity of ageing red cells in man. Br J Haematol 1967;13:665-678. 24 Magnani M, Piatti E, Serafini N, Palma F, Dacha M, Fomaini G. The age-dependent metabolic decline of the red blood cell. Mech Ageing Dev 1983;22:295-308. 25 Matovcik LM, Mentzer WC. The membrane of the human neonatal red cell. In: Schrier SL, ed. The red blood cell membrane. London: Saunders WB Co., 1985;203-221. 26 Stocchi V, Kolb N, Cucchiarini L, Segni M, Magnani M, Fomaini G. Adenine and pyridine nucleotides during rabbit reticulocyte maturation and cell ageing. Mech Ageing Dev 1987;39:29-44.
Clinica Chimica Acta, 189 (1990) 131-138 Elsevier
CCA 04735
The levels of adenine nucleotides and pyridine coenzymes in red blood cells from the newborn, determined simultaneously by HPLC Marilena Formato’,
Bruno Masala 1 and Giancarlo De Luca *
’ Institute of General Physiology and Biological Chemistry and 2 Institute of Applied Biology, University of Sassari, Sassari (Italy) (Received
11 February
1989; revision received
Key words: Newborn
RBC; Adenine
12 July 1989; accepted
nucleotide;
Pyridine
13 March
coenzyme;
1990)
HPLC
The concentrations of ATP, ADP, AMP; NADP and NADPH; NAD and NADH were determined in erythrocytes from healthy newborns and compared with those obtained in healthy adults. No significant differences were found for the adenine nucleotide concentrations, but NADH levels were reduced in newborn erythrocytes, with a consequent increase in the NAD/NADH ratio. Moreover, in newborn erythrocytes increased levels of NADP were observed, with a consequent increase in the NADP/NADPH ratio and a decrease in the NAD/NADP ratio. These results indicate the need to use reference values of the ratios NAD/NADH, NADP/NADPH and NAD/NADP from healthy newborns in the study of syndromes affecting the metabolism of erythrocytes in the newborn.
Introduction The determination of the levels of adenine nucleotides (ATP, ADP) (AMP) and those of pyridine nucleotides, both in the oxidized and in the reduced form (NAD, NADH; NADP, NADPH), gives important information on the energetic and redox metabolism of the cell. For instance, in normal human erythrocytes (RBC) the measurement of the energy charge is an index of cell integrity [l]. The adenine nucleotide pool is modified in RBC of subjects affected with different pathological conditions [2-61, and a decrease of ATP concentration was observed during cell ageing [7-131. The measurement of pyridine nucleotides and the determination of
Correspondence to: Prof. Bruno MasaIa, Istituto di Fisiologia di Sassari, Via Muroni 25, I-07100 Sassari, Italy.
0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
Generale
e Chin&a
B.V. (Biomedical
Division)
Biologica,
Universita
132
NAD/NADH and NADP/NADPH ratios indicate the efficiency of glycolysis and the pentose-phosphate shunt. Moreover, the NADP/NADPH ratio has been found to be significantly altered in RBC of patients affected with hemolytic anemias [14,15]. It is still not known whether these parameters vary with the age of the subject, although this information may be critical in the study of any pathological condition affecting the newborn. For this reason we determined the concentrations of adenine nucleotides and pyridine coenzymes in RBC from normal human newborns by means of a recently developed reversed-phase HPLC method, which allows their simult~eous dete~nation after alkaline extraction [16,17]. Materials and methods The blood samples were obtained by venepuncture from 16 healthy newborns (within 2 days of delivery), and from 13 healthy adults (with a mean age of 30 yr) and collected in EDTA as ~tico~~~t. Ethical approval for the blood samples was obtained from both parents of each of the newborns. A micromethod employing 75 x 1 mm i.d. capillary tubes was used for hematocrit estimation. Samples were subjected to alkaline extraction of nucleotides within approximately 1 h. Alkaline extraction of nucleotides from whole blood was carried out according to Stocchi et al. [16,17]: 1 ml of blood was added to 1 ml of ice%old 0.5 mol/l KOH to deproteinize and filtered by centrifugation at 2 500 x g x 10 min at 4OC on a CF SOA Amicon membrane (Amicon, Lexington, MA, USA). The clear ultrafiltered solution was adjusted to pH 6.5 with 1 mol/l KH,PO., and immediately used for HPLC analysis. Portions were also stored at - 80” C and analysed after different periods. Reproducible results were obtained in samples stored up to 3 mth. Nucleotides were separated and determined essentially as described by Stocchi et al. [16,17] by means of an HPLC system (LKB, Bromma, Sweden) equipped with a LKB LyChrosorb RP-18 column (250 X 4.6 mm i.d.), protected with a guard column filled with pellicular reverse-phase material. The injection volume of the alkaline-extracted sample was 20 ~1. The eluent consisted of 2 elements: 0.1 mol/l KH,PO,, pH 6.0 (buffer A), and 0.1 mol/l KH,PO, containing 2.5 mol/l CH,OH, pH 6.0 (buffer B). The most suitable gradient was 11 min at 100% buffer A, 8 min from 100% buffer A to 25% buffer B, 8 min from 25% to 90% buffer B, 6 min from 90% to 100% buffer B, and finally 6 min at 100% buffer B. The flow rate was 1 ml/mm at room temperature, and detection was at 254 nm. Identity of the peaks was confirmed by coelution with standards (Sigma, St. Louis, MO, USA). Quantitative dete~nations were performed by injecting standard solutions of known concentration. Concentration of adenine and pyridine nucleotides was expressed as mmol/I RBC, using the hematocrit to correct the whole blood volume. Energy charge (EC) value was determined according to the equation: EC = (ATP + 1/2ADP)/(ATP + ADP + AMP). An analysis of the marginal distribution of individual variables was performed before the calculation of mean, standard deviation and Student’s t test. The degree of conformity to the normal dist~bution was evaluated via beets and kurtosis coefficients. A symmetrizing transformation was applied to the varia-
133
bles showing a significant asymmetry. Student’s t test was used to determine the significance of differences between the mean of the two groups for each of the variables. LOO
0
10
20
30
40
TIME (min)
Fig. 1. Simultaneous reversed-phase HPLC separation of adenine nucleotides and pyridme coenzymes. A, Alkaline extract from human adult RBC; B, alkaline extract from human newborn RBC. Due to the low concentration of some nucleotides in erythrocyte specimens, the sensitivity of the U.V. detector was increased from 0.05 to 0.01 absorbance unit of full scale (A.U.F.S.) after elution of ATP and ADP.
134
Results
Figure 1 shows the separation of adenine nucleotides and pyridine coenzymes in RBC from adults and newborn infants. The concentrations of adenine nucleotides and pyridine coenzymes (together with the values of EC, NAD/NADH, NADP/NADPH and NAD/NADP ratios) in these subjects are shown in Tables I and II, respectively. A few variables (NADH, NAD/NADH, ADP in the case of adults; NADH, NAD/NADH, NADP/NADPH, ADP in the case of newborns) were asymmetric on the original scale, but did not show appreciable asymmetry once transformed on a logarithmic scale. Means and 95% reference limits for these variables were calculated on the transformed scale. The adult RBC concentration of both adenine and pyridine nucleotides, EC value, and the ratios of oxidized/reduced coenzymes were in agreement with those described previously [16,17]. Neither adenine nucleotide concentrations nor EC value obtained in neonatal RBC differ significantly from those of adults. On the contrary, neonatal NADH levels appeared to be reduced and in 6 out of the 16 newborns resulted undetectable, so precise determination was impossible. For this reason, we could not include these values in the relevant analysis, obtaining a mean value which resulted non-significantly different from that of adult RBC. Therefore, the actual mean value has to be certainly lower, with a consequent significant increase in the NAD/NADH ratio. Significantly increased levels of NADP and the NADP/NADPH ratio were observed in neonates. Consequently, the NAD/NADP ratio was lower in neonatal RBC. Discussion
The data obtained in this study shows that both adenine nucleotide concentrations and the energy charge in neonatal RBC do not differ significantly from those of adults. On the contrary, in newborn RBC the concentration of NADH was lower, and NAD/NADH ratio consequently higher. Since a good correlation has been re-
TABLE I Mean and 95% reference limits for the concentration of adenine nucleotides and energy charge value in adult and newborn red blood ceIIs
Adults (n =13) Newborns (n =16) t
ATP
ADP
AMP
E.C.
1530 (1160-l 900) 1516 (1168-l 864) -0.2 b
172.43 a (116.51-255.19) 145.5 a (104.30-203.00) - 2.52 b
16.10 (8.89-23.31) 16.75 (7.40-26.10) 0.41 b
0.940 (0.924-0.956) 0.946 (0.934-0.958) 2.41 b
Values are expressed as mmoI/l BBC. * Variable non nomiaBy distributed and transformed in logatithrns. b p > 0.01 non-significant.
135
I
136
ported between the value of NAD/NADH ratio and RBC ability to utilize glucose [18], our results might suggest a higher rate of the glycolytic pathway in the neonatal RBC. This interpretation is also sustained by the findings that the newborn RBC displays higher percentages of young cells than are found in adults [19,20], and that both anaerobic glycolysis [ll] and hexokinase activity [7,9,13,21-241 decrease during RBC ageing. Moreover, the NADP and NADPH determinations in neonates and adults showed significant differences. NADP concentrations were higher in neonates, whereas NADPH levels appeared to be slightly reduced, with a consequent marked increase in the NADP/NADPH ratio. Since NADPH is necessary for regeneration of reduced glutathione in the glutathione reductase reaction, the increase in NADP/NADPH ratio probably reflects a higher NADPH utilization in newborn red cells related to their increased susceptibility to oxidation [25]. The NAD/NADP ratio was also significantly lower in neonatal RBC. This is consistent with the recent report that the NAD/NADP ratio increases in rabbit RBC with ageing [26], since newborn red cells have higher percentage of young cells [19,20] and therefore a shorter life-span. The reason for the reduced life-span of neonatal RBC is not known. If membrane damage by free radicals is of major importance [25], the increased NADP concentration might promote the pentosephosphate shunt and thus provide some protection by increased reducing capacity. We might conclude that, in the study of erythrocyte metabolism in pathological conditions affecting the newborn, it is particularly important to use normal values of NAD/NADH, NADP/NADPH, and NAD/NADP ratios determined in healthy newborn, since they significantly differ from the corresponding values in adult RBC. Acknowledgements
This work was supported by funds from M.P.I. (40%), Italy. We are greatly indebted to Prof. L. Belardinelli, who kindly helped us in statistical analysis. References 1 Atkinson DE. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 1%8;7:4030-4034. 2 Valentine WN, Paglia DE, Tartagba AP, Gilsanz F. Hereditary hemolytic anemia with increased red ceil adenosine deaminase (45- to ‘IO-fold) and decreased adenosine triphosphate. Science 1977;195:783-785. 3 Ericson A, Niklasson F, de Verdier CH. A systematic study of nucleotide analysis of human erythrocytes using an anionic exchanger and HPLC. Chn Chim Acta 1977;127,47-59. 4 Staal GEJ, Jansen G, Ross D. Pyruvate kinase and the ‘High ATP syndrome’. J Clin Invest 1984;74:231-235. 5 Stocchi V, Magnani M, Cucchiarini L, NoveBi G, Dallapiccola B. Red blood ceil adenine nucleotides abnormalities in Down syndrome. Am J Med Gen 1985;20:131-135. 6 Novelh G, Stocchi V, Giannotti A, Magnani M, DaIlapiccola B. Increased erythrocyte adenosine deaminase activity without haemolytic anaemia. Hum Hered 1986;36:37-40. 7 Brewer GX General red ceII metabolism. In: Surgenor D MacN, ed. The red blood cell, Vol I. New York: Academic Press, 1974;387-433.
137 8 Liehtman MA. Does ATP decrease exponentially during red cell ageing? Nouv Rev Fr Hematol 1975;15:625-632. 9 Fomaini G, Magnani M, Dacha M, Bosst M, Stocchi V. Relationship between glucose phosphorylating activities and erythrocytes age. Mech Ageing Dev 1978;8:249-256. 10 Magnani M, Stocchi V, Dacha M, Canestrari F, Fomaini G. Hexokinase isozymic pattern during red cell ageing. FEBS Lett 1980;120:264-266. 11 Seaman C, Wyss S, Piomelli S. The decline in energetic metabolism with ageing of the erythrocyte and its relationship to cell. Am J Hematol 1980;8:31-42. 12 Stocchi V, Magnani M, Canestrari F, Dacha M, Fomaini G. Multiple forms of human red blood cell hexokinase. Preparation, characterization, and age dependence. J Biol Chem 1982;257:2357-2364. 13 Magnani M, Fazi A, Accorsi A, Stocchi V, Dacha M, Fomaini G. Regulatory properties of human erythrocyte hexokinase during cell ageing. Arch Biochem Biophys 1985;239:352-358. 14 Kirkman I-IN, Gaetani GD, Clemons EH, Mareni C. Red cell NADP and NADPH in glucose-6-phosphate dehydrogenase deficiency. J Clin Invest 1975;55:875-878. 15 Magnani M, Stocchi V, Canestrari F, et al. Redox and energetic state of red blood cells in G6PD deficiency, heterozygous beta-thalassemia and the combination of both. Acta Haematol 1986;75:211214. 16 Stocchi V, Cucchiarini L, Magnani M, Chiarantini L, Palma P, Crescentini G. Simultaneous extraction and reverse-phase high-performance chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal Biochem 1985;146:118-124. 17 Stocchi V, Cucchiarini L, Canestrari F, Piacentini P, Fomaini G. A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells. Anal Biochem 1987;167:181-191. 18 Stocchi V, Cucchiarini L, Magnani M, Fomaini G. Adenine and pyridine nucleotides in the erythrccyte of different mammalian species. Biochem Int 1987;14:1043-1053. 19 Masala B, Manta L, Format0 M, Pi10 G. A study of the switch of fetal hemoglobin in newborn erythrocyte fractionated by density gradient. Hemoglobin 1983;7:567-572. 20 Smith JB, Butzner JD, Wills MR, Sabio H, Savory J. Erythrocyte creature in cord blood. Ann Clin Lab Sci 1983;13:439-443. 21 Turner BM, Fischer RA, Harris H. The age related loss of activity of four enzymes in the human erythrocytes. Clin Chim Acta 1974;50:85-95. 22 Rogers PA, Fisher RA, Harris H. An examination of the age-related patterns of decay of the hexokinase of human red cells. Clin Chim Acta 1975;65:291-298. 23 Chapman RG, Shaumberg L. Glycolysis and glycolytic enzyme activity of ageing red cells in man. Br J Haematol 1967;13:665-678. 24 Magnani M, Piatti E, Serafini N, Palma F, Dacha M, Fomaini G. The age-dependent metabolic decline of the red blood cell. Mech Ageing Dev 1983;22:295-308. 25 Matovcik LM, Mentzer WC. The membrane of the human neonatal red cell. In: Schrier SL, ed. The red blood cell membrane. London: Saunders WB Co., 1985;203-221. 26 Stocchi V, Kolb N, Cucchiarini L, Segni M, Magnani M, Fomaini G. Adenine and pyridine nucleotides during rabbit reticulocyte maturation and cell ageing. Mech Ageing Dev 1987;39:29-44.