121
Clink Chimica Ada, 192 (1990) 121-132 Elsevier
CCA 04845
Erythrocyte nucleotide stability and plasma hypoxanthine concentrations: improved ATP stability with short-term storage at room temperature H.A.
Simmonds,
Purine Research Laboratories,
V. Micheli
*, P.M. Davies
Clinical Science Laboratories, London (UK)
and M.B. McBride
UMDS of Guy’s & St. Thomas’ Hospitals,
(Received 16 May 1990; revision received and accepted 13 August 1990)
Key work Erythrocyte nucleotide stability; Hypoxanthine; Inosine; Hypoxanthine-guanine phosphoribosyltransferase; Uridine diphosphate sugar; Phosphoribosylpyrophosphate synthetase; NAD; Nicotinamide; Heparinised blood
We have measured erythrocyte nucleotide concentrations at timed intervals over 24 h in heparinised blood stored at 4” C, room temperature, or 37 o C. The objective was to determine whether the grossly altered NAD concentrations found in the erythrocytes of patients with two different inherited purine disorders could be related to altered stability or turnover rates. An unexpected finding was the improved stability of all erythrocyte nucleotides in blood stored at room temperature compared with 4°C. Not only was the breakdown of ATP greater at 4” C compared with room temperature, higher hypoxanthine concentrations were present in the plasma associated with a fictitious increment in inosine. NAD and NADP, by contrast, showed remarkable stability in both control and patient erythrocytes, irrespective of their original value. Although these studies failed to establish an explanation for the altered NAD levels in the patients, the superior ATP stability in blood stored at room temperature in the erythrocytes from both patients and controls suggests that current practices of storing blood on ice for short-term studies require re-evaluation.
* Present address: Istituto di Chimica Biologica, Siena, Italy. Correspondence to: Dr. H.A. Simmonds, Purine Research Laboratories, Clinical Science Laboratories, Guy’s Tower (17/18 floor), Guy’s Hospital, London Bridge SE1 9RT, UK. 0009-8981/90/$03.50
Q 1990 Elsevier Science Publishers B.V. (Biomedical Division)
122
Introduction
Altered erythrocyte nucleotide patterns have been reported in patients with the X-linked disorder, hypoxanthine-guanine phosphoribosyltransferase (HGPRT: EC 2.4.2.8) deficiency and also in some patients with the other X-linked disorder PP-ribose-P synthetase (PPRPS: EC 2.7.6.1) superactivity [l]. Both disorders have a broad spectrum of clinical expression, being associated in their severest form with neurological deficits which include compulsive self-mutilation in HGPRT deficiency (21, and inherited nerve deafness in PPRPS super~ti~ty 131.Erythrocyte nucleotide patterns tend to reflect the clinical severity, the most marked alterations being the very high concentrations of the pyridine di-nucleotide NAD in the Lesch-Nyhan syndrome, as compared with the extremely low NAD values in the most severly affected patients with the synthetase mutant [l]. The instability of nucleotides in stored blood, particularly the adenine ribonucleotide ATP, is well known [1,4-131; nucleotides in different animal species are particularly labile compared with human cells [6]. Consequently, it was of interest to determine whether the grossly altered NAD concentrations in the above disorders could be related to either an altered stability or turnover rate. The recommended practice has been to store blood on ice [14-171 to minimise the fall in ATP concentrations, which also produces a spurious increment in plasma hypoxanthine 14-181. In this paper we have compared nucleotide stability in erythrocytes from patients with the above disorders and healthy controls over different periods of time and at three different storage temperatures. Subjects aud methods Patients and controls
Blood was obtained from seven healthy volunteers, two patients with adolescent gout and partial HGPRT deficiency [2] and a male patient and his heterozygous mother with PPRPS superactivity [3], during ~vestigation of current clinical status. One patient with the Lesch-Nyhan syndrome (complete HGPRT deficiency) was also studied. AlI three patients with HGPRT deficiency were being treated with allopurinol. Full clinical details have been published [1,3,22-233. The experimental design The experimental design included storing blood from patients and controls
at different temperatures over a range of times up to 24 h. For completeness, where sufficient sample was available, further measurements were made at 48 h and in one control after 120 h. Because of the small amount of blood available from the patient with the Lesch-Nyhan syndrome and the patient with the synthetase mutant, the studies were only carried out at 4O C and 37” C over 24 h. This was based on the assumption that these extremes would represent the most stable and unstable conditions, respectively.
123
sample preparation Venous blood was divided at the time of venipuncture into 2 ml sterile lithium heparin containers and mixed gently. Heparin was used because of the high UV absorbance of EDTA in the high performance liquid chromatography (HPLC) system used. One blood sample from each individual was centrifuged immediately at 1500 x g for 5 minutes at room temperature and the plasma and packed cells separated as rapidly as possible. The other samples were stored at either 4* C, room temperature (20-23* C) or 37” C respectively and then centrifuged as above and separated at the time intervals indicated, up to 24 h (and in a few instances 48-120 h). Nucleotide concentrations were then determined in erythrocytes, and nucleoside and base concentrations were determined in plasma as follows.
Nucleotide concentrations The packed cells in each instance were washed twice with isotonic sodium chloride (154 mmol/l) and the haematocrit recorded to determine the final packed cell volume and enable subsequent quantification of nucleotide concentration in pmol/l according to the method of Dean et al. 161. One volume of the packed washed erythrocytes (e.g. 100 ~1) was then added to two volumes (e.g. 200 ~1) of 0.6 mol/l trichloroacetic acid (TCA) in a 1.5 ml Eppendorf tube, using a positive displacement pipette, while mixing vigorously on a vortex mixer. After centrifugation at room temperature for 2 mm at 9000 X g in a Beckman microfuge the TCA was extracted immediately from the supematant using water-saturated diethyl-ether until the pH of the supematant was above 5.0 (4-5 extractions).
Quantification using HPLC Quantification using HPLC was carried out using a fully-automated Milhpore Waters System (Harrow, UK) as described [1,24]. 25 pl of extract was injected onto an anion exchange column (5 pm APS-Hypersil 25 cm x 4.9 mm) and the nucleotides separated using a linear phosphate buffer gradient elution system (buffer A: 5 mmol/l KH,PO, pH 265; buffer B: 0.5 mol/l KH,PO,Ji.O mol/l KCl, pH 3.5) at a flow rate of 1 ml/min with dual wavelength UV detection at 254 and 280 nm. Nucleotide concentrations were calculated according to Dean et al. [6], using a formula which takes into account the haematocrit plus the fact that the erythrocyte is approximately 2/3 water [24].
NucIeoside and base concentrations One volume of plasma (e.g. 250 ~1) from either fresh or stored blood separated at the time intervals indicated was precipitated with an equal volume of 0.6 mol/l TCA, centrifuged i~e~ately at room temperature for 2 mm at 9000 X g and the supematant extracted as above with diethyl-ether to a pH above 5.0. Nucleosides and bases in 50 ~1 of the resultant extract were also quantified by HPLC using a Hichrom 5 pm ODS column and the reversed phase system described 123,241.
124
Results
Effect of temperature and time on nucleotide levels The mean nucleotide concentrations in erythrocytes from the controls are compared (Fig. 1) with the results for the patients with partial HGPRT deficiency, the Lesch-Nyhan patient, and the individuals with the mutant PPRPS. The high NAD concentrations in the Lesch-Nyhan and partially HGPRT-deficient erythrocytes contrast with the low values in the PPRPS mutant and the intermediate levels. in his heterozygote mother [1,3].
Purine nucleotide concentrations Purine nucleotide concentrations in control cells stored at 4” C, room temperature or 37 o C for the time intervals indicated (Table I) overall showed a fall in ATP and GTP levels with time. The surprising finding, however, was that ATP showed greater stability in blood stored at room temperature than at 4 o C, the concentrations after 4 and 8 h (Table I) being little changed from control values, while the values after 24 h were still much higher at room temperature than at 4 o C or 37 o C in cells from patients and controls (Fig. 1). IMP levels increased under all conditions, but were highest at 37°C and 4°C. (After 48 h at room temperature a fall in ATP levels was accompanied by a rise in AMP levels with a further increment in IMP in the few subjects where sufficient blood was available - data not shown. Although these results may be complicated by many factors, the total adenine derived nucleotides - including IMP - were still well in excess of 70% of the sum at zero time.) In cells stored at 4O C, degradation of ATP and GTP was already evident after 4 h and the concentrations decreased steadily thereafter (Table I). By contrast ATP levels were highest initially after 4 h in cells stored at 37 o C associated with a fall in ADP (Table I), which probably relates to the glucose level. However, after 8 h at 37 o C total adenylates were similar to the concentrations at 4O C and after 24 h at 37°C both bis and &phosphates of ATP and GTP were low to barely detectable. (After 48 h, the value for total adenylates had fallen to 50% or below at 4’C and were 10% or less at 37” C, in contrast to the results described above at room temperature.)
The pyridine nucleotides The pyridine nucleotides NAD, NADP and UDPG, by contrast, were stable in control erythrocytes at any temperature or time of storage up to 24 h (Table I), and even over a 48 h period, as well as in a single study over 120 h (data not shown). The NAD concentrations in patients’ erythrocytes likewise showed little change with time, despite the high or low endogenous levels, respectively, in the HGPRT deficient and PPRPS mutant erythrocytes (Fig. 1). In the few individuals where the sample was sufficient to investigate the effect of storage for 48 h, cells from both patients and controls at 37 o C showed evidence of some haemolysis, accompanied by slightly lower NAD levels and the appearance of the NAD breakdown products, nicotinamide and ADP-ribose, in the plasma (data not shown). The important
ATIP ADP
ATP ADP
:
NAD NADP UDPC
bLTP ADP
AMP IMP NAD NADP UDPC
PPRPS mutant : patient a
HGPRT- : pticnt a
ATP ADP
AMP IMP NAD NADP UDPC
PPRPS mutant : paticnt b
HGPRT- : pdicnt b
in erythrocyte nucleotide concentrations (pmol/l) in heparinised blood after storage for 24 h at 4OC, room (A and B) with partial HGPRT deficiency (HGPRT-) compared with the control mean, and (lower panel) at of a Lesch-Nyhan patient plus the patient with the mutant PPRPS (a) and his heterozygote mother (b).
AMP IMP NAD NADP UDPC
Lcsch-Nyhan patient
AMP IMP
Inca”
Fig. 1. The upper panel indicates the change temperature (RT) or 37 o C from two patients 4O C and 37 o C only for the erythrocytes
600
200
600
l800
control
4 -C
24 h at 37 4:
24 h at
24 h at RT
fresh blood
E
557 (366-772) 999 (639-l 423) 49 (34-50)
946 (697-l 055) 1294 (1205-1401) 701 (665-745)
1057 (817-1300) 1282 (1122-1481) 1380 (1289-1481)
534)
178 (92-252) 217 (135-290) 45 (25-66)
234 (118-299) 121 (101-136) 286 (231-340)
;1?6-232) 202 (176-227) 105 (97-112)
162 (155-174)
ADP packed cells)
140 (32-208) 121 (44-175) 104 (75-149)
113 (22-168) 16 (12-19) 200 (137-262)
(O-8)
;:3-31) 3
14 (7-24)
16 (13-18) 20 (16-26) 13 (10-16)
;;7-27) 31 (28-34)
21 (14-34)
31 (29-32)
11 (8-18)
;!6-123) 27 (24-34) 13 (7-18)
GTP
AMP
&22)
14 (12-15) 14 (10-19)
17 (13-20) 10 (8-11) 14 (8-20)
17 (10-16) 15 (13-16) 12 (9-13)
13 (9-15)
GDP
:3:-74) 198 (80-299)
159 (72-270)
79 (75-89)
69 (60-76) 68 (60-74) 65 (60-71)
57 (54-61) 54 (52-58) 48 (43353)
57 (51-59) 56 (51-60) 55 (50-58)
70 (60-72) 69 (62-73) 69 (61-71)
56 (49-60) 63 (52-66) 57 (52-61)
56 (50-63)
NADP
22
67 (60-73) 70 (61-74) 70 (61-73)
64 (60-74)
NAD
(2-40)
7 (3-14)
(l-5) _
3
IMP
32 (29-38) 33 (29-39) 25 (19-29)
(27-39) 35 (29-40) 35 (31-41)
35
34 (30-38) 34 (30-38) 35 (30-39)
37 (32-43)
UDPG
The table lists the adenine and guanine nucleotide concentrations (pmol/l), as well as the NAD, NADP and UDPG concentrations, in the erythrocytes of healthy controls (mean plus the range) after storage of heparinised blood for 4, 8 and 24 hours at the three temperatures indicated, compared with the values at zero time. - = below the limits of detection.
37O
RT
24h 4O
37O
RT
8h 4O
37O
RT
4h 4O
1313 (1198-l
ATP (pmol/l
Erythrocyte nucleotides: stability with time
TABLE I
s
127
TABLE I1 Plasma purine levels in erytbrocyte stability experiments (pmol/l) UA
HX
299 (264-333)
1 (0.5-2.0)
298 (271-332) 301 (262-329) 291 (264-318)
20 (11-32) 13 (7-20) 10 (8-11)
300 (269-331) 303 (272-323) 293 (261-324)
38 (29-54)
309 (269-328) 291 (269-329) 288 (256-314)
87 (54-157) 52 (18-85) 454 (278-651)
X/G
0 time
4h
4OC RT 37” 8h
4O RT 37O 24h 4O RT 37O
HR
GR
_
_
_
_ ;2-6) _
_
_
_
_
16 (9-24)
_
_ ;:l-18) 35 (25-42)
1
1
_
_
1
58 (38-76) _
2
5
_
8 (2-15)
The table lists the mean concentrations of purines in the plasma (pmol/l) of healthy controls after separation from the erytbrocytes at the times and temperatures indicated in Table I. Ranges are given only for those values which varied significantly from the mean. The plasma uric acid (UA) showed remarkable stability with time. H = hypoxanthine, HR = inosine, G = guanine, GR = guanosine, X = xanthine. - = below the limits of detection.
finding was that despite the considerable variation in NAD concentrations in the patients’ erythrocytes compared with the controls, the stability with time and temperature showed a remarkable degree of comparability and the range of variation was less than 10% of the mean as noted for control erythrocytes (Table I).
Effect of temperature and time on nucleoside and base levels The nucleotide degradation products found in the plasma in control blood reflected the findings in the erythrocytes (Table II) and confirmed that blood stored at room temperature showed the smallest rise in nucleoside and base levels, the principal increase being in hypoxanthine, with the levels being much higher in blood stored at either 4O C or 37 o C. Curiously, at 4” C, inosine also accumulated at levels up to half that of hypoxanthine from 4 h onwards, suggesting some inhibition of PNP at this temperature. At 37” C hypoxanthine levels increased as anticipated from barely detectable at zero time to levels in the millimolar range at 48 h. Low
128
129
levels of ADP-ribose, nicotinamide and UMP were also detected in the plasma at 48 h as indicated above (data not shown). The nucleoside and base concentrations in the plasma of the different patient groups following storage of blood for 24 h at 4” C, room temperature (patients with partial HGPRT deficiency only) or 37 o C are also compared (Fig. 2) with the mean levels found in control plasma over the same period. The variation in concentration of these components in patients’ plasma likewise reflected the temperature of storage, with room temperature again showing the lowest increment in values compared with fresh blood and inosine being detected only in blood stored at 4 o C in all instances. It was noteworthy that the plasma uric acid showed remarkable stability with time and no adenosine accumulated under any conditions in the blood of either patients or controls. Very low levels of a component with the retention time and 280/254 ratio of xanthine were noted in control cells (X/G, Table II, Fig. 2) the basal levels being slightly higher in two of the HGPRT deficient subjects on allopurinol. The small increment in this component with storage was presumed to be due to guanine derived from GTP breakdown, since (a) these two compounds could not be separated by the HPLC system used; (b) the erythrocyte lacks both guanase and xanthine oxidase (c) the greatest increase in ‘xanthine’ occurred at 24 h and 37OC (except in the Lesch-Nyhan cells with low GTP levels [l]) and a slight increment in guanosine was also noted at 4°C (not shown).
Discussion Although, the present in vitro studies have failed to establish any biochemical basis in terms of altered NAD stability to explain the extremely low NAD concentrations noted in the erythrocytes of some patients with the severe form of the X-linked disorder PPRPS superactivity [1,3], compared with the very high levels found in the Lesch-Nyhan syndrome or partial HGPRT deficiency [1,2], nevertheless, the results have proved extremely interesting in terms of nucleotide stability. The lability of the adenine and guanine nucleotides contrasted sharply with the stability demonstrated for NAD in the human erythrocyte and confirmed the importance of evaluating nucleotide concentrations as soon as possible after venipuncture. The interesting and unexpected finding in these studies was the variation in the rate of nucleotide degradation depending on the temperature of blood storage, with the best stability being evident in blood stored at room temperature over a 24 h period. This was surprising since it has long been accepted practice, where immediate processing was impossible, that blood should be stored on ice [14-171 to avoid the spurious increment in plasma hypoxanthine resulting from ATP degradation [4,10-13,15-181. The greatest increment in plasma hypoxanthine in our studies was in blood stored at 4 o C. Conflicting results regarding the effect of temperature on erythrocyte nucleotide stability have been published previously by several groups, but the data refer to a limited number of subjects [8,15,16]. The present
130
results in this larger group consisting of both patients and controls accord with data pub~shed previously by Ericson and De Verdier IS]. The greater erythrocyte nucleotide stability in blood stored at room temperature in the present study was supported by the much higher increment in the plasma hypoxanthine at 4°C and 37°C. Additionally, blood stored at 4°C showed a fictitious increment in plasma inosine, which could explain why some workers consistently report inosine in plasma [11,27], and suggests that PNP is partially inhibited in erythrocytes at 4°C [28,29]. The raised IMP levels, together with the fact that no increment in adenosine was noted, confirms that the degradation of ATP normally proceeds predominantly via AMP to IMP in the erythrocyte [lo]. The finding that at room temperature, even after storage for 24 h and longer, over 70% of the original ATP content was still to be found within the cell, predo~n~tly as ADP and AMP, suggests that AMP deaminase was equally sensitive to storage temperature, being more active at 4O C or 37OC. In contrast to the other nucleotides, NAP) and NADP (and also the pyrimidine sugar UDPG) demonstrated remarkable stability, irrespective of their original value. The extraordinary stability of NAD in the erythrocytes contrasts with the rapid turnover reported for NAD in nucleated cells, which is seemingly a matter of hours [25]. The results suggest that either the erythrocyte lacks the enzymes capable of degrading these pyridine (and pyrimidine) nucleotides, that they are non-functional in the intact erythrocyte, or do not have access to these nucleotides because they are extensively protein bound. The slight decrease in NAD and UDPG afte 48 h at 37* C could be attributed to cell lysis because of the finding of low levels of ~cotina~de, ADP-ribose and UMP in the plasma at 37” C, together with the evidence of slight haemolysis. NAD gIycohydrolase is known to be an ectoenzyme 1261. It is thus likely that the enzyme degrading UDPG is also located on the external surface of the red cell. Although these short-term in vitro studies failed to establish an explanation for the abnormal NAD levels in the erythrocytes of patients with HGPRT deficiency or PP-ribose-P synthetase superactivity [1,3], the important and unexpected observation was the superior stability of all nucleotides evident in blood of both patients and controls stored at room temperature, associated with a much lower increment in plasma hypoxanthine. Since much interest currently attaches to the measurement of adenosine as well as hypoxanthine in different clinical situations [12,13,29-211, extreme care must obviously be taken regarding sample handling to ensure the validity of the results obtained. Our results at 4°C question the current practice of storing blood on ice. Comparative studies are thus necessary to determine whether storage on ice does provide the most reliable results. Acknowledgements
These studies were supported by the Arthritis and Rheumatism Council, the British Council and the Special Trustees of Guy’s Hospital. The advice and support of Professor C. Ricci, late director of the Istituto di Chimica Biologica is gratefully acknowledged.
131
References 1 Simmonds HA, Fairbanks LD, Morris GS, Webster DR, Harley EH. Altered erythrocyte nucleotide patterns are characteristic of inherited disorders of purine and pyrimidine metabolism. Clin Chim Acta 1988;171:197-210. 2 Kelley WN, Wyngaarden JB. Clinical syndromes associated with hypoxanthine-guanine phosphoribosyltransferase deficiency. In: Stanbury JB, Wyngaarden JM, Frederickson DS, Goldstein JL, Brown M, eds. The Metabolic Basis of Inherited Disease, New York: McGraw-Hill. 1983;ch 51:1115-1143. 3 Simmonds HA, Webster DR, Wilson J, Lingham S. An X-linked syndrome characterised by hyperuricaemia, deafness and neurodevelopmental abnormalities. Lancet 1982;2:68-70. 4 Jorgensen S. Breakdown of adenine and hypoxanthine nucleotides and nucleosides in human plasma. Acta Pharmacol Toxic01 1956;12:294-302. 5 Mills GC, Summers BL. The metabolism of nucleotides and other phosphate esters in erythrocytes during in vitro incubation at 37°C. Arch Biochem Biophys 1959;84:7-14. 6 Dean BM, Perrett D, Sensi M. Changes in nucleotide concentrations in the erythrocytes of man, rabbit and rat during short-term storage. Biochem biophys Res Commun 1978;80:147-154. 7 Bartlett GR. Metabolism of adenosine and deoxyadenosine by stored human red cells. Adv Exp Med Biol 1980;122A:409-414. 8 Ericson A, De Verdier CH. Specimen handling for the assay of adenylates and glyceraldehyde 2,3-bisphosphate in erythrocytes. Stand J Clin Lab Invest 1981;41:361-366. 9 Boulieu R, Bory C, Gonnet C. Liquid chromatographic measurement of purine nucleotides in blood cells. Clin Chem 1985;31:727-731. 10 Bontemps F, Van den Berghe G, Hers HG. Pathways of adenine catabolism in erythrocytes. J Clin Invest 1986;77:824-830. 11 Stocchi V, Cucchiarini L, Canestrari F, Placentini MP, 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;167:181-190. 12 Harkness RA. Hypoxanthine, xanthine and uridine in body fluids, indicators of ATP depletion. J Chromatog 1988;429:255-278. 13 Saugstad OD. Hypoxanthine as an indicator of hypoxia: its role in health and disease through free radical production. Pediatric Res 1988;23:143-150. 14 Goldfinger S, Klinenberg JR, Seegmiller JE. The renal excretion of oxypurines. J Clin Invest 1965;44:623-628. 15 Bakaria M, Brown PR. Investigation of clinical methodology for sample collection and processing prior to the reversed-phase liquid chromatographic determination of UV-absorbing plasma constituents. Anal Biochem 1982;120:25-37. 16 Boulieu R, Bory C, Boltassat P, Gonrtet C. Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte and urine samples from healthy subjects: the problem of hypoxanthine evolution as a function of time. Anal Biochem 1983;129:398404. 17 Puig JG, Mateos FA, Jiminez ML, Ramos TH. Renal excretion of xanthine and hypoxanthine in primary gout. Am J Med 1988;85:533-537. 18 Wung WE, Howell SB. Simultaneous liquid chromatography of 5fluorouraci1, uridine, hypoxanthine, xanthine, uric acid, allopurinol and oxipurinol in plasma. Clin Chem 1980;26:1704-1708. I9 Valen PA, Nakayama DA, Veum JA, Wortman RL. Myoadenylate deaminase deficiency: diagnosis by ischaemic forearm exercise testing and plasma purine measurements. Pediatric Res 1985;19:779786. 20 Granger DN, Hollwarth NE, Parks DA. Ischaemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiol Stand 1986;548:47-63. 21 Sinkeler SPT, Joosten EMG, Wevers RA, et al. Ischaemic exercise test in myoadenylate deaminase deficiency and McArdle’s disease: measurement of plasma adenosine, inosine and hypoxanthine. Clin Sci 1986;70:399-410.
132 22 Simmonds HA, Cameron JS, Barratt TM, Dillon MJ, Meadow SR, Trompeter RS. Purine enzyme defects as a cause of acute renal failure in childhood. Pediatric Nephrol 1989;3:433-437. 23 Morris GS, Simmonds HA, Davies PM. Use of biological fluids for the rapid diagnosis of potentially lethal inherited disorders of human purine and pyrimidine metabolism. Biomed Chromatogr 1986;1:109-118. 24 Simmonds HA, Duley JA, Davies PM. Analysis of purines and pyrimidines in blood, urine, and other physiological fluids. In: Hommes FA, ed. Techniques in diagnostic human biochemical genetics: a laboratory manual. New York: Wiley-Liss, 1990;397-424, ch 25. 25 Rechsteiner MC, Hillyard D, Olivera BM. Magnitude and significance of NAD turnover in the human cell line D/98/AH2. Nature 1976;259:695,696. 26 Goodman SI, Wyatt RJ, Trepel JB, Neckers LM. NAD ~ycohydrol~e: enzyme characteristics using intact rn~~~ erythroeytes. Comp B&hem Physiol 1982;71:333-336. 27 Mateos FA, Puig JG, Jiminez ML, Fox IH. Hereditary xanthinuria: evidence for enhanced hypoxanthine salvage. J Clin Invest 1987;79:847-852. 28 Kremtsky TA, Elion GB, Henderson AM, Hitchings GH. Inhibition of human purine nucleoside phosphorylase. Studies with intact erythrocytes and the purified enzyme. J Biol Chem 1968;243:2876-2881. 29 Kim BK, Cha S, Parkes RE. Purine nucleoside phosphorylase from human erythrocytes: purification and properties. J Biol Chem 1968;243:1763-1770.
Clink Chimica Ada, 192 (1990) 121-132 Elsevier
CCA 04845
Erythrocyte nucleotide stability and plasma hypoxanthine concentrations: improved ATP stability with short-term storage at room temperature H.A.
Simmonds,
Purine Research Laboratories,
V. Micheli
*, P.M. Davies
Clinical Science Laboratories, London (UK)
and M.B. McBride
UMDS of Guy’s & St. Thomas’ Hospitals,
(Received 16 May 1990; revision received and accepted 13 August 1990)
Key work Erythrocyte nucleotide stability; Hypoxanthine; Inosine; Hypoxanthine-guanine phosphoribosyltransferase; Uridine diphosphate sugar; Phosphoribosylpyrophosphate synthetase; NAD; Nicotinamide; Heparinised blood
We have measured erythrocyte nucleotide concentrations at timed intervals over 24 h in heparinised blood stored at 4” C, room temperature, or 37 o C. The objective was to determine whether the grossly altered NAD concentrations found in the erythrocytes of patients with two different inherited purine disorders could be related to altered stability or turnover rates. An unexpected finding was the improved stability of all erythrocyte nucleotides in blood stored at room temperature compared with 4°C. Not only was the breakdown of ATP greater at 4” C compared with room temperature, higher hypoxanthine concentrations were present in the plasma associated with a fictitious increment in inosine. NAD and NADP, by contrast, showed remarkable stability in both control and patient erythrocytes, irrespective of their original value. Although these studies failed to establish an explanation for the altered NAD levels in the patients, the superior ATP stability in blood stored at room temperature in the erythrocytes from both patients and controls suggests that current practices of storing blood on ice for short-term studies require re-evaluation.
* Present address: Istituto di Chimica Biologica, Siena, Italy. Correspondence to: Dr. H.A. Simmonds, Purine Research Laboratories, Clinical Science Laboratories, Guy’s Tower (17/18 floor), Guy’s Hospital, London Bridge SE1 9RT, UK. 0009-8981/90/$03.50
Q 1990 Elsevier Science Publishers B.V. (Biomedical Division)
122
Introduction
Altered erythrocyte nucleotide patterns have been reported in patients with the X-linked disorder, hypoxanthine-guanine phosphoribosyltransferase (HGPRT: EC 2.4.2.8) deficiency and also in some patients with the other X-linked disorder PP-ribose-P synthetase (PPRPS: EC 2.7.6.1) superactivity [l]. Both disorders have a broad spectrum of clinical expression, being associated in their severest form with neurological deficits which include compulsive self-mutilation in HGPRT deficiency (21, and inherited nerve deafness in PPRPS super~ti~ty 131.Erythrocyte nucleotide patterns tend to reflect the clinical severity, the most marked alterations being the very high concentrations of the pyridine di-nucleotide NAD in the Lesch-Nyhan syndrome, as compared with the extremely low NAD values in the most severly affected patients with the synthetase mutant [l]. The instability of nucleotides in stored blood, particularly the adenine ribonucleotide ATP, is well known [1,4-131; nucleotides in different animal species are particularly labile compared with human cells [6]. Consequently, it was of interest to determine whether the grossly altered NAD concentrations in the above disorders could be related to either an altered stability or turnover rate. The recommended practice has been to store blood on ice [14-171 to minimise the fall in ATP concentrations, which also produces a spurious increment in plasma hypoxanthine 14-181. In this paper we have compared nucleotide stability in erythrocytes from patients with the above disorders and healthy controls over different periods of time and at three different storage temperatures. Subjects aud methods Patients and controls
Blood was obtained from seven healthy volunteers, two patients with adolescent gout and partial HGPRT deficiency [2] and a male patient and his heterozygous mother with PPRPS superactivity [3], during ~vestigation of current clinical status. One patient with the Lesch-Nyhan syndrome (complete HGPRT deficiency) was also studied. AlI three patients with HGPRT deficiency were being treated with allopurinol. Full clinical details have been published [1,3,22-233. The experimental design The experimental design included storing blood from patients and controls
at different temperatures over a range of times up to 24 h. For completeness, where sufficient sample was available, further measurements were made at 48 h and in one control after 120 h. Because of the small amount of blood available from the patient with the Lesch-Nyhan syndrome and the patient with the synthetase mutant, the studies were only carried out at 4O C and 37” C over 24 h. This was based on the assumption that these extremes would represent the most stable and unstable conditions, respectively.
123
sample preparation Venous blood was divided at the time of venipuncture into 2 ml sterile lithium heparin containers and mixed gently. Heparin was used because of the high UV absorbance of EDTA in the high performance liquid chromatography (HPLC) system used. One blood sample from each individual was centrifuged immediately at 1500 x g for 5 minutes at room temperature and the plasma and packed cells separated as rapidly as possible. The other samples were stored at either 4* C, room temperature (20-23* C) or 37” C respectively and then centrifuged as above and separated at the time intervals indicated, up to 24 h (and in a few instances 48-120 h). Nucleotide concentrations were then determined in erythrocytes, and nucleoside and base concentrations were determined in plasma as follows.
Nucleotide concentrations The packed cells in each instance were washed twice with isotonic sodium chloride (154 mmol/l) and the haematocrit recorded to determine the final packed cell volume and enable subsequent quantification of nucleotide concentration in pmol/l according to the method of Dean et al. 161. One volume of the packed washed erythrocytes (e.g. 100 ~1) was then added to two volumes (e.g. 200 ~1) of 0.6 mol/l trichloroacetic acid (TCA) in a 1.5 ml Eppendorf tube, using a positive displacement pipette, while mixing vigorously on a vortex mixer. After centrifugation at room temperature for 2 mm at 9000 X g in a Beckman microfuge the TCA was extracted immediately from the supematant using water-saturated diethyl-ether until the pH of the supematant was above 5.0 (4-5 extractions).
Quantification using HPLC Quantification using HPLC was carried out using a fully-automated Milhpore Waters System (Harrow, UK) as described [1,24]. 25 pl of extract was injected onto an anion exchange column (5 pm APS-Hypersil 25 cm x 4.9 mm) and the nucleotides separated using a linear phosphate buffer gradient elution system (buffer A: 5 mmol/l KH,PO, pH 265; buffer B: 0.5 mol/l KH,PO,Ji.O mol/l KCl, pH 3.5) at a flow rate of 1 ml/min with dual wavelength UV detection at 254 and 280 nm. Nucleotide concentrations were calculated according to Dean et al. [6], using a formula which takes into account the haematocrit plus the fact that the erythrocyte is approximately 2/3 water [24].
NucIeoside and base concentrations One volume of plasma (e.g. 250 ~1) from either fresh or stored blood separated at the time intervals indicated was precipitated with an equal volume of 0.6 mol/l TCA, centrifuged i~e~ately at room temperature for 2 mm at 9000 X g and the supematant extracted as above with diethyl-ether to a pH above 5.0. Nucleosides and bases in 50 ~1 of the resultant extract were also quantified by HPLC using a Hichrom 5 pm ODS column and the reversed phase system described 123,241.
124
Results
Effect of temperature and time on nucleotide levels The mean nucleotide concentrations in erythrocytes from the controls are compared (Fig. 1) with the results for the patients with partial HGPRT deficiency, the Lesch-Nyhan patient, and the individuals with the mutant PPRPS. The high NAD concentrations in the Lesch-Nyhan and partially HGPRT-deficient erythrocytes contrast with the low values in the PPRPS mutant and the intermediate levels. in his heterozygote mother [1,3].
Purine nucleotide concentrations Purine nucleotide concentrations in control cells stored at 4” C, room temperature or 37 o C for the time intervals indicated (Table I) overall showed a fall in ATP and GTP levels with time. The surprising finding, however, was that ATP showed greater stability in blood stored at room temperature than at 4 o C, the concentrations after 4 and 8 h (Table I) being little changed from control values, while the values after 24 h were still much higher at room temperature than at 4 o C or 37 o C in cells from patients and controls (Fig. 1). IMP levels increased under all conditions, but were highest at 37°C and 4°C. (After 48 h at room temperature a fall in ATP levels was accompanied by a rise in AMP levels with a further increment in IMP in the few subjects where sufficient blood was available - data not shown. Although these results may be complicated by many factors, the total adenine derived nucleotides - including IMP - were still well in excess of 70% of the sum at zero time.) In cells stored at 4O C, degradation of ATP and GTP was already evident after 4 h and the concentrations decreased steadily thereafter (Table I). By contrast ATP levels were highest initially after 4 h in cells stored at 37 o C associated with a fall in ADP (Table I), which probably relates to the glucose level. However, after 8 h at 37 o C total adenylates were similar to the concentrations at 4O C and after 24 h at 37°C both bis and &phosphates of ATP and GTP were low to barely detectable. (After 48 h, the value for total adenylates had fallen to 50% or below at 4’C and were 10% or less at 37” C, in contrast to the results described above at room temperature.)
The pyridine nucleotides The pyridine nucleotides NAD, NADP and UDPG, by contrast, were stable in control erythrocytes at any temperature or time of storage up to 24 h (Table I), and even over a 48 h period, as well as in a single study over 120 h (data not shown). The NAD concentrations in patients’ erythrocytes likewise showed little change with time, despite the high or low endogenous levels, respectively, in the HGPRT deficient and PPRPS mutant erythrocytes (Fig. 1). In the few individuals where the sample was sufficient to investigate the effect of storage for 48 h, cells from both patients and controls at 37 o C showed evidence of some haemolysis, accompanied by slightly lower NAD levels and the appearance of the NAD breakdown products, nicotinamide and ADP-ribose, in the plasma (data not shown). The important
ATIP ADP
ATP ADP
:
NAD NADP UDPC
bLTP ADP
AMP IMP NAD NADP UDPC
PPRPS mutant : patient a
HGPRT- : pticnt a
ATP ADP
AMP IMP NAD NADP UDPC
PPRPS mutant : paticnt b
HGPRT- : pdicnt b
in erythrocyte nucleotide concentrations (pmol/l) in heparinised blood after storage for 24 h at 4OC, room (A and B) with partial HGPRT deficiency (HGPRT-) compared with the control mean, and (lower panel) at of a Lesch-Nyhan patient plus the patient with the mutant PPRPS (a) and his heterozygote mother (b).
AMP IMP NAD NADP UDPC
Lcsch-Nyhan patient
AMP IMP
Inca”
Fig. 1. The upper panel indicates the change temperature (RT) or 37 o C from two patients 4O C and 37 o C only for the erythrocytes
600
200
600
l800
control
4 -C
24 h at 37 4:
24 h at
24 h at RT
fresh blood
E
557 (366-772) 999 (639-l 423) 49 (34-50)
946 (697-l 055) 1294 (1205-1401) 701 (665-745)
1057 (817-1300) 1282 (1122-1481) 1380 (1289-1481)
534)
178 (92-252) 217 (135-290) 45 (25-66)
234 (118-299) 121 (101-136) 286 (231-340)
;1?6-232) 202 (176-227) 105 (97-112)
162 (155-174)
ADP packed cells)
140 (32-208) 121 (44-175) 104 (75-149)
113 (22-168) 16 (12-19) 200 (137-262)
(O-8)
;:3-31) 3
14 (7-24)
16 (13-18) 20 (16-26) 13 (10-16)
;;7-27) 31 (28-34)
21 (14-34)
31 (29-32)
11 (8-18)
;!6-123) 27 (24-34) 13 (7-18)
GTP
AMP
&22)
14 (12-15) 14 (10-19)
17 (13-20) 10 (8-11) 14 (8-20)
17 (10-16) 15 (13-16) 12 (9-13)
13 (9-15)
GDP
:3:-74) 198 (80-299)
159 (72-270)
79 (75-89)
69 (60-76) 68 (60-74) 65 (60-71)
57 (54-61) 54 (52-58) 48 (43353)
57 (51-59) 56 (51-60) 55 (50-58)
70 (60-72) 69 (62-73) 69 (61-71)
56 (49-60) 63 (52-66) 57 (52-61)
56 (50-63)
NADP
22
67 (60-73) 70 (61-74) 70 (61-73)
64 (60-74)
NAD
(2-40)
7 (3-14)
(l-5) _
3
IMP
32 (29-38) 33 (29-39) 25 (19-29)
(27-39) 35 (29-40) 35 (31-41)
35
34 (30-38) 34 (30-38) 35 (30-39)
37 (32-43)
UDPG
The table lists the adenine and guanine nucleotide concentrations (pmol/l), as well as the NAD, NADP and UDPG concentrations, in the erythrocytes of healthy controls (mean plus the range) after storage of heparinised blood for 4, 8 and 24 hours at the three temperatures indicated, compared with the values at zero time. - = below the limits of detection.
37O
RT
24h 4O
37O
RT
8h 4O
37O
RT
4h 4O
1313 (1198-l
ATP (pmol/l
Erythrocyte nucleotides: stability with time
TABLE I
s
127
TABLE I1 Plasma purine levels in erytbrocyte stability experiments (pmol/l) UA
HX
299 (264-333)
1 (0.5-2.0)
298 (271-332) 301 (262-329) 291 (264-318)
20 (11-32) 13 (7-20) 10 (8-11)
300 (269-331) 303 (272-323) 293 (261-324)
38 (29-54)
309 (269-328) 291 (269-329) 288 (256-314)
87 (54-157) 52 (18-85) 454 (278-651)
X/G
0 time
4h
4OC RT 37” 8h
4O RT 37O 24h 4O RT 37O
HR
GR
_
_
_
_ ;2-6) _
_
_
_
_
16 (9-24)
_
_ ;:l-18) 35 (25-42)
1
1
_
_
1
58 (38-76) _
2
5
_
8 (2-15)
The table lists the mean concentrations of purines in the plasma (pmol/l) of healthy controls after separation from the erytbrocytes at the times and temperatures indicated in Table I. Ranges are given only for those values which varied significantly from the mean. The plasma uric acid (UA) showed remarkable stability with time. H = hypoxanthine, HR = inosine, G = guanine, GR = guanosine, X = xanthine. - = below the limits of detection.
finding was that despite the considerable variation in NAD concentrations in the patients’ erythrocytes compared with the controls, the stability with time and temperature showed a remarkable degree of comparability and the range of variation was less than 10% of the mean as noted for control erythrocytes (Table I).
Effect of temperature and time on nucleoside and base levels The nucleotide degradation products found in the plasma in control blood reflected the findings in the erythrocytes (Table II) and confirmed that blood stored at room temperature showed the smallest rise in nucleoside and base levels, the principal increase being in hypoxanthine, with the levels being much higher in blood stored at either 4O C or 37 o C. Curiously, at 4” C, inosine also accumulated at levels up to half that of hypoxanthine from 4 h onwards, suggesting some inhibition of PNP at this temperature. At 37” C hypoxanthine levels increased as anticipated from barely detectable at zero time to levels in the millimolar range at 48 h. Low
128
129
levels of ADP-ribose, nicotinamide and UMP were also detected in the plasma at 48 h as indicated above (data not shown). The nucleoside and base concentrations in the plasma of the different patient groups following storage of blood for 24 h at 4” C, room temperature (patients with partial HGPRT deficiency only) or 37 o C are also compared (Fig. 2) with the mean levels found in control plasma over the same period. The variation in concentration of these components in patients’ plasma likewise reflected the temperature of storage, with room temperature again showing the lowest increment in values compared with fresh blood and inosine being detected only in blood stored at 4 o C in all instances. It was noteworthy that the plasma uric acid showed remarkable stability with time and no adenosine accumulated under any conditions in the blood of either patients or controls. Very low levels of a component with the retention time and 280/254 ratio of xanthine were noted in control cells (X/G, Table II, Fig. 2) the basal levels being slightly higher in two of the HGPRT deficient subjects on allopurinol. The small increment in this component with storage was presumed to be due to guanine derived from GTP breakdown, since (a) these two compounds could not be separated by the HPLC system used; (b) the erythrocyte lacks both guanase and xanthine oxidase (c) the greatest increase in ‘xanthine’ occurred at 24 h and 37OC (except in the Lesch-Nyhan cells with low GTP levels [l]) and a slight increment in guanosine was also noted at 4°C (not shown).
Discussion Although, the present in vitro studies have failed to establish any biochemical basis in terms of altered NAD stability to explain the extremely low NAD concentrations noted in the erythrocytes of some patients with the severe form of the X-linked disorder PPRPS superactivity [1,3], compared with the very high levels found in the Lesch-Nyhan syndrome or partial HGPRT deficiency [1,2], nevertheless, the results have proved extremely interesting in terms of nucleotide stability. The lability of the adenine and guanine nucleotides contrasted sharply with the stability demonstrated for NAD in the human erythrocyte and confirmed the importance of evaluating nucleotide concentrations as soon as possible after venipuncture. The interesting and unexpected finding in these studies was the variation in the rate of nucleotide degradation depending on the temperature of blood storage, with the best stability being evident in blood stored at room temperature over a 24 h period. This was surprising since it has long been accepted practice, where immediate processing was impossible, that blood should be stored on ice [14-171 to avoid the spurious increment in plasma hypoxanthine resulting from ATP degradation [4,10-13,15-181. The greatest increment in plasma hypoxanthine in our studies was in blood stored at 4 o C. Conflicting results regarding the effect of temperature on erythrocyte nucleotide stability have been published previously by several groups, but the data refer to a limited number of subjects [8,15,16]. The present
130
results in this larger group consisting of both patients and controls accord with data pub~shed previously by Ericson and De Verdier IS]. The greater erythrocyte nucleotide stability in blood stored at room temperature in the present study was supported by the much higher increment in the plasma hypoxanthine at 4°C and 37°C. Additionally, blood stored at 4°C showed a fictitious increment in plasma inosine, which could explain why some workers consistently report inosine in plasma [11,27], and suggests that PNP is partially inhibited in erythrocytes at 4°C [28,29]. The raised IMP levels, together with the fact that no increment in adenosine was noted, confirms that the degradation of ATP normally proceeds predominantly via AMP to IMP in the erythrocyte [lo]. The finding that at room temperature, even after storage for 24 h and longer, over 70% of the original ATP content was still to be found within the cell, predo~n~tly as ADP and AMP, suggests that AMP deaminase was equally sensitive to storage temperature, being more active at 4O C or 37OC. In contrast to the other nucleotides, NAP) and NADP (and also the pyrimidine sugar UDPG) demonstrated remarkable stability, irrespective of their original value. The extraordinary stability of NAD in the erythrocytes contrasts with the rapid turnover reported for NAD in nucleated cells, which is seemingly a matter of hours [25]. The results suggest that either the erythrocyte lacks the enzymes capable of degrading these pyridine (and pyrimidine) nucleotides, that they are non-functional in the intact erythrocyte, or do not have access to these nucleotides because they are extensively protein bound. The slight decrease in NAD and UDPG afte 48 h at 37* C could be attributed to cell lysis because of the finding of low levels of ~cotina~de, ADP-ribose and UMP in the plasma at 37” C, together with the evidence of slight haemolysis. NAD gIycohydrolase is known to be an ectoenzyme 1261. It is thus likely that the enzyme degrading UDPG is also located on the external surface of the red cell. Although these short-term in vitro studies failed to establish an explanation for the abnormal NAD levels in the erythrocytes of patients with HGPRT deficiency or PP-ribose-P synthetase superactivity [1,3], the important and unexpected observation was the superior stability of all nucleotides evident in blood of both patients and controls stored at room temperature, associated with a much lower increment in plasma hypoxanthine. Since much interest currently attaches to the measurement of adenosine as well as hypoxanthine in different clinical situations [12,13,29-211, extreme care must obviously be taken regarding sample handling to ensure the validity of the results obtained. Our results at 4°C question the current practice of storing blood on ice. Comparative studies are thus necessary to determine whether storage on ice does provide the most reliable results. Acknowledgements
These studies were supported by the Arthritis and Rheumatism Council, the British Council and the Special Trustees of Guy’s Hospital. The advice and support of Professor C. Ricci, late director of the Istituto di Chimica Biologica is gratefully acknowledged.
131
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132 22 Simmonds HA, Cameron JS, Barratt TM, Dillon MJ, Meadow SR, Trompeter RS. Purine enzyme defects as a cause of acute renal failure in childhood. Pediatric Nephrol 1989;3:433-437. 23 Morris GS, Simmonds HA, Davies PM. Use of biological fluids for the rapid diagnosis of potentially lethal inherited disorders of human purine and pyrimidine metabolism. Biomed Chromatogr 1986;1:109-118. 24 Simmonds HA, Duley JA, Davies PM. Analysis of purines and pyrimidines in blood, urine, and other physiological fluids. In: Hommes FA, ed. Techniques in diagnostic human biochemical genetics: a laboratory manual. New York: Wiley-Liss, 1990;397-424, ch 25. 25 Rechsteiner MC, Hillyard D, Olivera BM. Magnitude and significance of NAD turnover in the human cell line D/98/AH2. Nature 1976;259:695,696. 26 Goodman SI, Wyatt RJ, Trepel JB, Neckers LM. NAD ~ycohydrol~e: enzyme characteristics using intact rn~~~ erythroeytes. Comp B&hem Physiol 1982;71:333-336. 27 Mateos FA, Puig JG, Jiminez ML, Fox IH. Hereditary xanthinuria: evidence for enhanced hypoxanthine salvage. J Clin Invest 1987;79:847-852. 28 Kremtsky TA, Elion GB, Henderson AM, Hitchings GH. Inhibition of human purine nucleoside phosphorylase. Studies with intact erythrocytes and the purified enzyme. J Biol Chem 1968;243:2876-2881. 29 Kim BK, Cha S, Parkes RE. Purine nucleoside phosphorylase from human erythrocytes: purification and properties. J Biol Chem 1968;243:1763-1770.
