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[CANCER RESEARCH 44, 3144-3148. July 1984]
Hypoxanthine Concentrations in Normal Subjects and Patients with SolidTumors and Leukemia1
Wally E. Wung and Stephen B. Howell2
Department of Medicine, the Cancer Center, and the General Clinical Research Center, University of California, San Diego, La Jolla, California 92093
ABSTRACT
Mean plasma hypoxanthine (Hyp) concentrations determinedby high-pressure liquid chromatography were 0.56 MM (range,0.2 to 1.9 MM)in 16 normal subjects, 0.68 MM(range, 0.1 to 1.1MM)in 10 untreated acute leukemic subjects, and 0.89 MM(range,0.3 to 2.6 MM)in 14 solid tumor patients. Despite large differencesin Hyp concentration between patients, every 4-hr sampling,
indicated that diurnal variation in individual patients was small(maximum, 2.3-fold). While the mean plasma and malignanteffusion Hyp concentrations did not differ significantly, bonemarrow plasma Hyp concentration averaged 4.0-fold greater
than that of simultaneously drawn venous plasma. Allopurinol300 mg p.o. caused a mean 1.5-fold increase in plasma Hyp
within 3 hr. In 17 patients with acute leukemia, treatment withallopurinol at 300 mg daily plus initiation of chemotherapy causeda mean 7-fdd increase in plasma Hyp to 4.6 MM(range, 1 to 12MM).The ability of Hyp to modulate the toxicity of antimetabolitesaffecting purine synthesis (6-diazao-5-oxonorleucine, 6-methyl-mercaptopurine riboside, 6-mercaptopurine, and 6-thioguanine)was determined in vitro using human B-lymphoblast (WI-L2) andpromyelocytic leukemia (HL-60) cell lines. Hyp permitted growth
of both cell lines in the presence of clinically achievable concentrations of all 4 drugs, but the initial culture concentrations ofHyp required were above those found in patients. Since Hypwas consumed rapidly during the culture period, the averageHyp concentrations required for the protection of cells wereactually much lower. We conclude that, in patients with acuteleukemia receiving allopurinol during chemotherapy, plasma Hypconcentrations are significantly elevated; the potential for antagonism of antimetabolite activity is uncertain.
INTRODUCTION
Antimetabolites which interfere with de novo purine synthesisconstitute an important part of the armamentarium of drugsavailable for the treatment of leukemia and other cancers (4, 15,26, 36). However, not all patients respond equally well to thesedrugs, and some eventually become refractory to the therapy.Resistance to purine analogues has been attributed to the deficiency of enzymes necessary to convert them to their nucleotideforms (7, 31). Other mechanisms have been observed in experimental tumors, such as increased degradation of drugs andinability of resistant cells to convert the ribonucleotide analoguesto the deoxyribonucleotide analogue (10, 29).
This study was designed to examine the possibility that in-
1This work was supported by Grants CA23100 and CA23334 from the National
Cancer Institute and by the University of California, San Diego General ClinicalResearch Center, NIH, Division of Research Resource (Grant RR00827). Thisresearch was conducted in part by the Clayton Foundation for Research, CaliforniaDivision.
2Clayton Foundation Investigator. To whom requests for reprints should be
addressed.Received April 10, 1981 ¡accepted April 2, 1984.
creased plasma Hyp3 concentrations during treatment may also
be a mechanism of resistance to antimetabolites acting in thepurine biosynthetic pathways. Extracellular Hyp is important forthe activity of purine antimetabolites in 2 aspects: it can beutilized to restore purine nucleotide pools in cells the de novosynthesis of which has been blocked, and it potentially maycompete with some of these antimetabolites for transport intothe cell or for the enzymes necessary to convert the drugs totheir active nucleotides (9).
Using a newly developed high-pressure liquid Chromatographie
method (43), we have measured plasma Hyp in groups of normalsubjects, patients with solid tumors, and patients with acuteleukemia. Frequent blood samples were obtained from somepatients to assess diurnal variation, and Hyp concentration inbone marrow and malignant effusions was compared to that ofvenous plasma to determine the distribution of Hyp in differentbody compartments. We also examined the effect on plasmaHyp of treatment with HPP alone or the combination of HPP andchemotherapy. Finally, the effect of Hyp at concentrations encompassing the range observed in vivo was tested for its abilityto modulate the toxicity of de novo purine synthesis inhibitorstoward the HL-60 promyelocytic leukemia and the WI-L2 lym-
phoblast human cell lines.
MATERIALS AND METHODS
Subjects and Patients. Blood samples were obtained from 5 groupsof subjects and patients for Hyp measurement: (a) normal laboratorypersonnel; (b) patients with acute lymphocytic leukemia or acute nonlym-
phocytic leukemia, either untreated or in relapse; (c) patients with advanced solid tumors of many types; (d) patients with malignant pleuralor peritoneal effusions due to solid tumor involvement; and (e) patientswith acute leukemia undergoing initial induction chemotherapy for eitheruntreated or relapsed disease. No restrictions were placed on diet oractivity in any group of subjects or patients, and samples were drawn atrandom times of day. For all groups except the normal subjects andacute leukemic subjects receiving induction chemotherapy, blood samples were obtained at least 3 weeks following any prior therapy and at atime when no toxicity attributable to prior therapy was evident.
High-Pressure Liquid Chromatography. A detailed procedure for
simultaneous measurement of plasma Hyp, uric acid, HPP, and oxipurinolwas developed in this laboratory and has been published elsewhere (43).Delay in separation of plasma from the formed elements of blood hasbeen shown to increase plasma Hyp. Therefore, samples were collectedin prechilled heparinized tubes, and plasma was separated immediatelyby centrifugation, precipitated with 0.1 volume of 4.4 N HCIO<, andneutralized with Alamine/Freon, as described by Khym (21). A high-
pressure liquid Chromatographie system (Waters Associates, Milford,MA) with a reverse-phase C,fi-^Bondapak column was used in this study.
Peaks were identified by their retention times and their absorbance ratiosat 254 and 280 nm. An aliquot of 20-/il sample was analyzed isocratically
'The abbreviations used are: Hyp, hypoxanthine; DON, 6-diazo-5-oxonorteu-cine; HPP, 4-hydroxy-3,4-pyrazotopyrimidine (allopurinol); HGPRT, hypoxanthine-guanine phosphoribosyltransferase; 6-MP, 6-mercaptopurine; MMPR, 6-methyl-mercaptopurine riboside; 6-TG. 6-thioguanine.
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Plasma Hyp and Antimetabolite Activity
using phosphate eluent (50 HIM KH2PO4, pH 4.60). The limit of detectionfor all compounds of interest was 0.1 MM.
Cell Culture and Growth Studies. A human B-lymphoblast (WI-L2)and a human promyelocytic leukemia (HL-60) cell line were used todetermine the growth-inhibitory effect of various concentrations of anti-
metabolites and the blockade of this effect by Hyp in vitro. These cellswere grown in RPMI 1640 medium (Cell Culture Facility, University ofCalifornia, San Diego, La Jolla, CA) supplemented with 10% fetal calfserum (Irvine Scientific, Irvine, CA), 1% glutamine, and 1% antibiotics-
antimycotics mixture (Grand Island Biological Co., Grand Island, NY).Growth assays were performed in LJnbro multiwell tissue culture plates(Flow Laboratories, Inc., McLean, VA). The tog-phase cells were washed
with RPM11640 medium to remove any Hyp in the growth medium. Hypand antimetabolites (6-MP, 6-TG, MMPR, and DON) were prepared in
the same medium, except that fetal calf serum was replaced with bovineserum albumin (5 mg/ml) or 10% charcoal-treated fetal calf serum. Cellswere cultured at 105/ml in triplicate with 1 to 100 MMHyp and 1, 4, and10 MM of each antimetabolite at 37° for 3 days under 4% CO2. The
number of cells was counted electronically using a Model ZB CoulterCounter, and the percentage of control growth in antimetabolite-treatedcultures was calculated by comparison with cells growing in Hyp-freeand antimetabolite-free medium.
RESULTS
Means and Ranges of Plasma Hyp. Previous reports haveindicated that, in acute leukemia, both the production and plasmaconcentration of uric acid were increased (29). Similar changesin plasma Hyp, which is the precursor of uric acid, have not beenreported. A comparison of plasma Hyp concentrations in randomly collected blood samples from 16 normal subjects, 10untreated acute leukemic subjects, and 14 untreated solid tumorpatients is summarized in Chart 1. The geometric mean plasmaHyp concentration was 0.56 MM(range, 0.2 to 1.9 MM)in normalsubjects, 0.68 MM (range, 0.1 to 1.1 MM) in acute leukemicsubjects, and 0.89 MM (range, 0.3 to 2.6 MM) in solid tumorpatients, and the differences between these groups were notsignificant. Included in Chart 1 are measurements made onmalignant ascites or pleural effusions from solid tumor patients.The geometric mean effusion Hyp concentration was 0.4 MM(range, 0.1 to 1.0 MM),about one-half of that in plasma of solid
tumor patients.Mean plasma uric acid concentration in these acute leukemic
4xlO~~
g5
IxlO,-6
o.
IxlO,-7
ft
T
NORMAL LEUKEMIC SOLID MALIGNANTSUBJECTS PATIENTS TUMOR EFFUSIONS
PATIENTS
Chart 1. Scattergram of plasma Hyp concentrations in normal subjects (n =16), acute leukemic subjects (n - 10), and solid tumor patients (n = 14). Hypconcentrations in malignant effusions (n = 9) are also included. Bars, geometric
means.
IO'6
ia,-70600 1200 1800 2400
TIME OF DAY
Chart 2. Diurnal variations of plasma Hyp concentration in 4 untreated solidtumor patients. No restrictions were placed on the patients' diets or activities.
patients was 410 MM(range, 110 to 610 MM)(data not shown),which is 1.5-fold greater than the mean of 276 MM(range, 210
to 340 MM)found in the normal subjects; plasma uric acid in thesolid tumor patients was not significantly different from that innormal subjects (mean, 255 MM;range, 150 to 440 MM).
Diurnal Variation of Plasma Hyp. Four solid tumor patientswere studied for diurnal variation of plasma Hyp. None of thesepatients was receiving chemotherapy or HPP at the time ofsampling. Blood was collected at intervals of 4 hr for a period of24 hr. No restriction was placed on food intake or physicalactivity during the sampling time. Chart 2 shows that, in each ofthese 4 patients, plasma Hyp concentration remained withinrelatively narrow limits over 24 hr compared to the wider rangefound between patients. The average variation in plasma Hypconcentration over 24 hr was 1.9-fold, and the range was 1.4-to 2.3-fold. The variation could not be related to meals, activity,
or sleep.Effects of HPP and Chemotherapy on Plasma Hyp. The
effect of a single dose of 300 mg of HPP p.o. on plasma Hypconcentration and the pharmacokinetics of HPP were examinedin the same 4 patients in whom diurnal variation was also studied.Chart 3 shows the plasma concentrations of Hyp, HPP, andoxypurinol, the major metabolite of HPP, during the 8 hr followingHPP ingestion. In 3 of 4 patients, plasma HPP peaked at 2 hrand then fell rapidly, with an elimination half-life of 55 to 65 min.
In the fourth patient, gastrointestinal absorption of HPP wasdelayed. Maximum plasma oxypurinol was reached about 2 hrafter HPP ingestion, and it averaged 35 MM(range, 21 to 60 MM).This level remained steady for each patient over the next 6 hr.Other investigators have also reported a long half-life of 14 to
20 hr for oxypurinol (3, 23). Plasma Hyp concentration increasedto a mean of 1.35 MM(range, 0.55 to 2.50 MM)during the 6-hrperiod following HPP ingestion. There was a 1.5-fold increase
over the mean Hyp concentration during the preceding 24 hrbefore the administration of HPP.
Plasma Hyp was measured before and then again 48 hr afterthe initiation of treatment with HPP, daunorubicin, and 1-ß-o-
arabinofuranosylcytosine on 6 courses in 5 patients with acuteleukemia (Chart 4). In addition, individual nonpaired measurements were made either before or 48 hr after initiation of treatment on 11 courses given to 6 other acute leukemic subjects.Whereas a single dose of HPP alone did not substantially increase plasma Hyp, the combination of HPP plus the initiation ofchemotherapy did. Considering only the 6 paired observations,the plasma Hyp concentration increased by a mean of 8.6-fold
(p < 0.01 ; f test for paired observations). When all measurements
JULY 1984 3145
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IV.E. Wung and S. B. Howe//
10-4rOXIPURINOL
V* \
01 2345678
HYPOXANTHINE
HOURS
Chart 3. Plasma HPP ( ), oxypurinol ( ), and Hyp concentration timecurves obtained in the same patients as in Chart 2 following the p.o. ingestion of a300-mg dose of HPP.
were included, the plasma Hyp concentration was 7-fold higher
at 24 hr, with a mean of 4.6 pM (range 0.9 to 12.0 /UM).A linearrelationship was observed between plasma Hyp and oxypurinolconcentrations measured in the same samples (r = 0.71 ; p <
0.01).Hyp Concentration in Bone Marrow. Hyp concentration in
bone marrow was compared with venous blood plasma in 9 solidtumor patients, and the results are shown in Chart 5. In allpatients, the marrow plasma Hyp concentrations were consistently greater than those of simultaneously drawn venous plasma.The mean difference between marrow plasma and venousplasma was 5.7 pu (p < 0.01 ; f test for paired observations).The possibility that this difference was due to hemolysis inmarrow specimens was excluded by the finding that there wasno increase in Hyp concentration in marrow plasma when RBCwere added and subjected to mechanical destruction.
Effect of Hyp on Antimetabolite Activity. Both WI-L2 andHL-60 cells are normally grown in medium containing 10% fetal
calf serum, which itself contributes large amounts of Hyp to theculture. In order to avoid this problem, WI-L2 cells were grownin purine-free RPMI 1640 medium containing bovine serum al
bumin (5 mg/ml), transferrin (35 pg/m\), insulin (5 pg/m\), 2.5 nwselenium, and 20 pM ethanolamine. HL-60 cells were grown inRPM11640 medium containing charcoal-stripped fetal calf serumat a final concentration of 10%. The fetal calf serum containedless than 1 pM Hyp after treatment with charcoal (0.5 g per 10ml of serum) as determined by high-pressure liquid chromatog-
raphy. Both media were supplemented with known concentra
tions of Hyp in experimental cultures.Chart 6 shows the effect of initial Hyp concentration between
1 and 100 ^M on the proliferation of WI-L2 and HL-60 cells inthe presence of 6-MP, 6-TG, MMPR, and DON. Antimetabolite
concentrations from 1 to 10 ^M were chosen as representativeof the concentrations of these drugs that can be achievedclinically (25, 27). Increasing the Hyp concentration 10-fold from
1 to 10 pM resulted in increased growth at all 3 concentrationsof all of the drugs; however, the magnitude of this effect wasnot large. A further 10-fold increase in initial Hyp concentration
increased growth substantially, even in cultures treated with thehighest antimetabolite concentrations, to 50% or more of thegrowth occurring in non-drug-treated control cultures for both
cell lines. Thus, Hyp antagonized the antiproliferative effect of all4 drugs, but a 50% reduction in antimetabolite effect requiredHyp concentration above those observed even in patients withacute leukemia receiving HPP and induction therapy who had amarked increase in Hyp production.
It is important to emphasize that the data depicted in Chart 6
I0~5r
O
gI-zLuO
S 10-6LU OZ J
Xl-
lo_
IO',-7
PRIOR TOTREATMENT
48HR AFTERSTART OF CHEMOTHERAPY AND HPP
Chart 4. Plasma Hyp concentrations in acute leukemic patients before and 48hr after the administration of HPP and combined chemotherapy consisting ofdoxorubicin and 1-/3-D-arabinofuranosylcytosine. HPP was given in daily doses of
300 mg p.o. during chemotherapy. Measurements made on the same patients areconnected by a solid line; bars, geometric means.
1-5
is1°•*-/ *
10,-6
PLASMA BONE MARROW
Chart 5. Comparison between venous plasma and bone marrow plasma Hypconcentrations in 9 solid tumor patients. Venous blood was collected at the momentof bone marrow aspiration; plasma was recovered from the 2-ml marrow aspirateby centrifugation. Measurements made on the same patients are connected by asolid line.
3146 CANCER RESEARCH VOL. 44
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Plasma Hyp and Antimetabolite Activity
WI-L2
DON
HL-60
DONDISCUSSION
IO"6 IO"5 IO"4 IO"6 IO"5 IO"4
HYPOXANTHINE CONCENTRATIONmol/liter
Chart 6. Effects of Hyp on the degree of growth inhibition produced by purineantimetaboliteson WI-L2 and HL-60cells during a 3-day culture. The antirnetaboliteconcentrations were irr" (A), 4 x 10"»(•),and KT5 (x) M.
Table 1Hyp concentration in cultures of WI-L2and HL-60 cells
Finalconcentration(pufConcentration0-M)1.0
10100Medium
alone1.0
10.598Medium
plusWI-L2ceUs"<0.1
<0.118Medium
plusHL-60cells'<0.1
1.169
8 After 3 days of culture.0 Finalcell density was 1.5 x KP/ml.c Final cell density was 0.9 x to'/mi.
are based on the initial concentration of Hyp in the culture. Table1 indicates that, while the concentration of Hyp remained stablein medium to which no cells were added, both WI-L2 and HL-60cells consumed much of the Hyp present during the 3-day period
of growth. Thus, the average concentration of Hyp was significantly lower than was the initial concentration, and this wasmore marked in cultures with the lower starting Hyp concentrations. The impact of considering average rather than initial Hypconcentrations on the curves depicted in Chart 6 would be toflatten their slopes somewhat but, on the other hand, it indicatesthat average Hyp concentrations even lower than 10 fiM werecapable of affecting purine antirnetabolite activity.
Little information is available on the concentration of Hyp invarious human body fluids under physiological conditions, andthe accuracy of concentrations reported previously has nowbeen called into question by the finding that special handling ofspecimens is required to avoid leakage of Hyp from RBC andplatelets (43). The release of Hyp during clotting probably accounts for the fact that the mean plasma Hyp values reportedhere are 10-fold less than are those reported for human serum
by other investigators (20, 33, 34).Although there was a wide variation in plasma Hyp concentra
tions measured at random times in individual subjects, a comparison of mean values in normal subjects, solid tumor patients,and acute leukemics revealed no significant differences amongthese groups. In addition, in individual subjects, plasma Hypconcentration varied by an average of only 1.9-fold during a 24-hr period. These observations suggest that plasma Hyp may beregulated by metabolic controls which must affect either theentrance of Hyp into the plasma or its clearance or both. Ourmeasurements provide some information on how changes in theproduction or clearance of Hyp influence plasma Hyp concentration. The finding of similar mean plasma Hyp concentrations inleukemic and normal subjects suggests that the increased production in leukemia (30) does not exceed the clearance capacityof xanthine oxidase or urinary excretion (8, 9, 14, 30). Undernormal circumstances, the clearance of Hyp via xanthine oxidaseis less significant than is that by other mechanisms, since inhibition of xanthine oxidase by HPP caused only a 1.5-fold increase
in Hyp concentration. However, when HPP was combined withincreased Hyp production resulting from initiation of chemotherapy in patients with acute leukemia, there was a 7-fold increase
in plasma Hyp concentration. Thus, under conditions of increased catabolic flux of purines, the activity of xanthine oxidasedoes have a very significant effect on plasma Hyp, and this wasreflected by the good correlation between plasma Hyp andoxyourinol, which is the active metabolite of HPP present inhighest concentration in plasma after administration of HPP (14,16).
The finding of a mean 4.0-fold higher concentration of Hyp in
bone marrow than in venous plasma is of interest for severalreasons. First, it suggests that, in contrast to liver, which clearslarge amounts of Hyp from plasma on a single pass (32), bonemarrow is a net producer of Hyp. A number of investigators haveconcluded that bone marrow cells and gastrointestinal mucosacells have a very limited capacity to carry out de novo purinesynthesis, and they depend on preformed purine in plasma forsynthesis of purine nucleotides via the salvage pathway (1, 24,28, 32, 39, 40). However, Hyp itself is an important regulator ofpurine synthesis (17-19, 36, 38), and concentrations as low as
1 /im are sufficient to decrease de novo synthesis in fibroblasts(38) and marrow (22). All of the previous studies on de novopurine synthesis in marrow cells were performed under conditions in which the concentration of Hyp in the cell environmentwas probably quite high (1,24,28,36,39,40). Thus, rather thanreflecting an inability of marrow cells to synthesize purine denovo, the limited incorporation of [14C]formate used to measure
the activity of this pathway may be due to the inhibitory actionof the high local concentration of Hyp. If the rapid cell proliferationin the gut is also accompanied by high local concentrations ofHyp, then the hypothesis that gut cells cannot produce purines
JULY 1984 3147
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W.E. WungandS. B. Howell
via théde novo route may also be incorrect (28).Several investigators have reported previously that Hyp can
reverse the cytotoxicity of MMPR (2, 37). Our finding that Hypreduces the activity of 6-MP, 6-TG, MMPR, and DON indicatesthat the effect is not merely due to competition for HGPRT orfor transport into the cell, since MMPR is phosphorylated byadenosine kinase rather than HGPRT (5, 35) and since DONdoes not require HGPRT for activation. The results of this studyshow that the concentrations of plasma Hyp in patients receivingHPP and chemotherapy are elevated into a range which is justbelow that which begins to interfere with the cytotoxic activityof clinically relevant concentrations of purine antimetabolitesagainst human leukemiacells in vitro. It is important to emphasizethat the curves in Chart 6 were constructed using the concentrations of Hyp present at the beginning of culture. Hyp concentration decreased rapidly as cells grew and, at the end of the 3-dayculture period, there was practically no Hyp left in the mediumoriginally containing 10 MMHyp, and there was a 30 to 60%reduction of Hyp in the cultures originally containing a 100 J/Mconcentration. Thus, no matter what the mechanism of the Hypeffect, the degrees of modulation depicted in Chart 6 wereactually achieved with average Hyp concentrations that wereconsiderably below the initial concentrations indicated on theabscissa. Although the growth conditions of leukemia cells Invitro are quite different than those in vivo, this raises the possibility that the 7-fold increase in plasma Hyp that accompaniedinitiation of chemotherapy in acute leukemic patients receivingHPP may be sufficient to reduce the effectiveness of the anti-purine antimetabolites during induction therapy. The increase inHyp also provides a potential explanation of the observation inrabbits that, although HPP can block the catabolism of 6-MP(11-13) and increase the area under the plasma concentrationtimes time curve (41), it does not increase the toxicity of 6-MP(42). Additional studies are required to evaluate completely therole of Hyp and HPP in modulating the activity 6-MP and theother antipurine antimetabolites in humans (6) and to validatethe extrapolation that we have made between in vitro resultsand in vivo observations.
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3148 CANCER RESEARCH VOL. 44
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