Stimulation
of Human Purine Synthesis by Fructose Infusion
Kari 0. Raivio, Michael A. Becker, Laurence J. Meyer, Martin George
Nuki, and J. Edwin
In order to clarify the mechanism of hyperuricemia and hyperuricosuria resulting from rapid infusion of fructose in man, the effects of an intravenous infusion of 125200 g of fructose given over 3-4 hr on the rate of purine synthesis de novo was measured in one individual with osteoarthritis and four patients with gout. The incorporation of l-“C glycine into urinary uric acid was measured, and the pool size and turnover of urate were assessed by renal excretion of simultaneously administered 15N urate. Fructose caused an expansion of body urate pool in all subjects, while urate turnover was increased in four. The rate of incorporation of “C glycine into urinary uric acid corrected for extrarenal disposal was increased in all cases (21%430%). In two patients, rates of incorporation of “C glycine into urinary creatinine were increased by 10% and 1 1 %, while
De Novo L. Greene,
Seegmiller
rates of incorporation into uric acid were increased 84% and 159%, respectively, as a result of fructose infusion. Specific enhancement of the rate of purine synthesis de novo was suggested by these findings. The rate of infusion appeared more important than total dose in determining the magnitude of this effect. Whether the increased rate of purine synthesis was a result of direct stimulation by a fructose metabolite or was secondary to fructoseinduced purine nucleotide depletion is uncertain, since the kinetics of glycine incorporation were consistent with either mechanism. Erythrocyte PP-ribose-P concentrations, however, were diminished during infusion rather than increased as might be expected if fructose infusion stimulated purine synthesis by increasing availability of this regulatory substrate.
I
NTRAVENOUS ADMINISTRATION of D-fructose produces increases in serum urate concentration and in urinary uric acid excretion in several mammalian species.lm3 A rapid breakdown of liver adenine nucleotides related to the rapid phosphorylation of fructose has been postulated to account for these effects,4m6 and decreases in liver adenine nucleotides have been measured during fructose loading the rat4,6v7and in man.* In a recent report, EmmersonY has assessed the effects of orally-administered fructose on the rate of incorporation of 14C glycine into urinary uric acid in man and has suggested an increased rate of purine synthesis de novo during fructose feeding. The purpose of the present study was to investigate whether the hyperuricemia and hyperuricosuria associated with intravenous infusion of fructose involves an increased rate of
From rhe Division of Rheumatology, Department of Medicine. University oj’ Califbrnia at San Diego and San Diego Velerans Administration Hospital. La Jolla, Calif: Receivedforpublication November8. 1974. Supported in part bv grants A M-13622, AM-05646, and MG-I 7702 of the National Instirures oJ Health and grants from the United States Veterans Administration. the Arthritis Foundation. the National Generics Foundation, and the National Foundation. Dr. Ravio was a fellow of the Arthritis Foundation and Dr. Nuki was a Merck Fellow in Clinical Pharmacology. Reprint requests should be addressed to Dr. J. Edwin Segmiller. Department of Medicine, M-013, University of Calvbrnia. San Diego, La Jolla, Cali/: 92037 .=I1975 bv Grune & Stratton. Inc.
Meiobolism,Vol.
24, No. 7(July), 1975
661
49
15
51
43
E. S.
SW.
T. B.
H. B.
values
57
KM.
Normal
w
Subject
Age
normal
excessive
of hip
increased synthetase
PP-ribose-P activity 10
uric acid production;
excessive
activity”
Gout;
PP-ribose-P
increased
synthetase
production;
excessive
uric-acid
Gout,
production
uric-acid
Gout,
production
uric-acid
Gout;
Osteoarthritis
Diagnosis
4
7
7
10
4
(days)
of study
Duration
210
510
510
580
550
mg/kg
10 min.
Initial
2630
1405
1405
1640
1560
Total
Dose of fructose
9.9
9.4
9.2
7.8
4.2
mg%
3.0-7.0
Baseline
Plasma
Urote
11.5
12.0
11.0
9.0
5.1
Infusion
During
413
980
1405
650
490
470
f
75 SD”
1019
1616
032
008
507
hr
Infusion
Hours of
First 24
Uric Acid
mgf24
Baseline
Urinarv
1650
15-25
1550
1725
1660
2060
mg/kg
1670
24 hr
1606
1608
1680
2140
hr
Infusion
Hours of
First 24
Creatininc
mg/24
Baseline
Urinary
Table 1. Clinical Summary of Study Patients and Effects of Fructose Infusion on Measurements of Purine Metabolism
2.8
5.0
5.1
3.0
3.2
2.7
f
nmoles/ml
Baseline
Eryihrocyie
0.5
SD”
4.5
4.0
2.8
2.8
2.2
pocked cells
Infusion
PP-ribose-P
PURINE
purine effect.
863
SYNTHESIS
nucleotide
synthesis
and, if possible,
MATERIALS
to define the mechanism
of such an
AND METHODS
Five male subjects were studied after each was given a full explanation of the implications of the studies and informed consent was obtained. Clinical characteristics of these individuals are summarized in Table 1. Renal function, as measured by creatinine clearance, was normal in all patients as was erythrocyte hypoxanthine-guanine-phosphoribosyltransferase activity.” Two patients had excessive activity of PP-ribose-P synthetase to account for their purine overproduction.” All were hospitalized on a metabolic ward and received no radiographic contrast materials or medications other than colchicine during the course of the studies. After a minimum of 5 days on a 2600 cal purine-free diet with I g protein/kg body weight, a control isotope study was carried out, followed at an interval of IO-14 days by a second isotope study in association with an infusion of fructose. The rate of purine synthesis de novo was estimated in each individual by measurement of the incorporation of 1-14C glycine (SrCi, 18.75 mCi/mmole, New England Nuclear Corporation) into urinary uric acid for periods ranging from 4 to IO days after oral administration of were corrected for extrathe isotope with breakfast milk. Values for 14C glycine incorporation renal disposal of urate as determined by simultaneous intravenous injection of lithium 1,3-“N urate (20 mg, 46.3 atom % excess, Isomet Corporation) in SY:, glucose.” Urine was collected in two l2-hr samples during the first day of each study and in 24-hr samples thereafter and was stored at room temperature with 3 ml of toluene as preservative. After analysis of urine samples for uric acidI and creatinine13 concentrations, uric acid was isolated and its content of 14C I4 and 15N Is was determined. Body urate pool and turnover, as well as corrected cumulative incorporation of label from glycine, were calculated as previously described.” On the morning of fructose infusion, oral intake of 250 ml of water every 30 min was begun I hr prior to infusion and continued during its course. Intravenous infusion of fructose was begun at the time of oral isotope administration. D-fructose, loo/, solution in water (Abbott Laboratories, Chicago), was administered as a rapid infusion of 450 ml over IO min followed by a 4-hr infusion at the rate of 200 ml/hr. Patient H.B. received a higher total dose of fructose but at a less-rapid rate during the initial IO min of infusion: 500 ml of the fructose solution over 30 min, followed by 500 ml over the next 60 min, and 1000 ml over the following 90 min. In all cases, except patient H.B., the dose of fructose during the initial 10 min of infusion exceeded 0.5 g/kg (Table I). a dose consistently associated epigastric discomfort during
with a hyperuricemic effect2 All subjects complained of transient the rapid injection of fructose, but no alarming objective signs de-
veloped. This symptom has previously been observed in conjunction with fructose administration.” Patients were allowed their regularly scheduled purine-free breakfasts during the infusion. In order to correct the isotopic data obtained in the fructose-infusion experiments for residual ‘sotope enrichment from the preceding control studies, the specific radioactivity of the urinary uric acid isolated throughout the control study was plotted as a function of time, and the values obtained by extrapolating the curve were subtracted from the 14C enrichments determined in the subsequent study. The validity and reproducibility of this method was studied by performance of two successive control studies at an interval of IO days in another individual. Using the method of correction described here, agreement to within 1% was found in this patient between the two values of corrected 14C glycine incorporation into urinary uric acid over 7 days. During the course of fructose infusion, blood samples for assay of plasma uric acid” and erythrocyte 5-phosphoribosyl I-pyrophosphate (PP-ribose-P)” concentrations were obtained at 0, IO, 30, 45, 60, 120. 180, and 240 min.
RESULTS
In agreement with earlier studies,” all subjects showed an increase (mean 22:,;,, range 167~;~28%) in plasma uric acid concentration with a peak at 30-120 min after the start of the fructose infusion (Table 1). Urinary uric acid excre-
1202
2321
2502
5. w. Control
Fructose
Fructose
H. B. Control
Fructose
T. 8. Control
0.48
1.34
0.56
1317
2815
0.72
0.65
2412
3585
0.46
0.47
0.54
1164
2518
Fructose
0.60
0.89
E. s. Control
2509
Fructose
Subject
R.M. Control
Turnover Rate K pools/day
1576
1770
2337
1736
1150
1100
1200
628
1505
1069
TUVlOVl?~ KA mg/day
Effects of Fructose infusion on Incorpomtion
Urote Pool Sire A mg
Table 2.
867
980
1439
1405
690
650
550
490
510
470
Average Daily Uric Acid Excretion E mg/day
of Isotopically
labeled
55
55
62
a0
60
59
46
78
35
44
TWnOVW Excreted E/KA %
40
57
60
78
47
50
51
62
41
44
Uric Acid
0.42
0.48
3.90
1.17
0.41
0.24
0.65
0.30
0.25
0.08
1.02
0.84
6.50
1.50
0.88
0.48
1.27
0.49
0.62
0.18
Administered Glycine Uncorrected Corrected G G/U % %
Cumulative Recovery of Isotope in Urinary Uric Acid
into Urinary
Administered Uric Acid U %
Uric Acid and Glycine
E
PURINE
865
SYNTHESIS
tion during the first 24 hr after beginning infusion was also increased; the range of this increase was 4%-65x above control values for excretion, and the mean increase was 27%. In contrast, a simultaneous decrease ranging between 87; and 21% with a mean of 14% was observed for erythrocyte PP-ribose-P concentration in all patients. The magnitude of this decrease was greatest between 30 and 90 min of infusion and was sustained through the remainder of the infusion. The changes in plasma urate and erythrocyte PP-ribose-P concentrations were transient and returned to baseline within 24 hr, while urinary uric acid excretion remained increased up to 48 hr. The results of the isotope studies are summarized in Table 2. Fructose infusion might be expected to disturb the steady state of urate pool size and turnover rate and thus render inaccurate the interpretation of measurements of these based on isotopic uric-acid injection. However, for each patient, semilog plot of uric acid isotope concentration versus time yielded a linear relationship as exemplified for patient T. B. in Fig. 1. Correlation coefficients for the least squares fit line relating log of uric-acid isotopic concentration to time varied from 0.92 to 0.99 among the five patients. Extrapolation of this line to time 0 was therefore used to estimate pool size, and turnover rate was estimated from
-2
78 SLOPE=-0652 DAYS-’ IA’JERCEPJ= -I 35 = ln 0 259
-3
In1
0
I
2
3 TIME
4
5
6
7
(days)
Fig. 1. Decline in isotope content ( 1) of urinary uric acid in patient T. B. ofter fructose infusion. Pool size was estimated from extrapolation of experimental line to time 0, and turnover rate was estimated from the slope of the line.
866
RAM0
ET Al.
the slope of the line. In each patient, fructose caused an increase in the urate pool which more than doubled in three cases. The turnover rate of the urate pool decreased in four of the subjects, raising the possibility that fructoseinduced lacticacidemia was impairing renal secretion of uric acid as previously described.” However, in agreement with a previous study,’ no consistent decrease in uric-acid clearance (during or after infusion) could be demonstrated, and turnover of urate was actually increased in all four subjects given an initial rapid rate of infusion. In addition, the cumulative recovery of administered “N urate in the urine was decreased by as much as 30% following fructose infusion, an observation difficult to explain on the basis of the transient lactic acidosis resulting from acute fructose loading. (In patient T. B., serial measurements of blood lactate during infusion showed a four-fold increase in lactate concentration to 33 mg/lOO ml at 90 min with a return to baseline value by 240 min.) Cumulative incorporation of 14Cglycine into urinary uric acid was markedly increased (range: 21%-430x) by fructose infusion in all cases, suggesting an enhanced rate of purine synthesis de novo (Table 2). (The magnitude of increase in incorporation was relatively little influenced by the length of the study, from 4 to 10 days.) An alternative explanation for the increased incorporation of 14C glycine into urinary uric acid is that fructose infusion diminished the endogenous glycine pools of tissues responsible for purine-nucleotide synthesis. This possibility was investigated by comparing cumulative 14Cglycine incorporation into urinary creatinine during the control and fructose studies of two patients (S. W. and E. S.). Creatinine was purified by the Benedict method” and isotopic enrichment was measured by liquid scintillation counting at 77% efficiency in Aquasol phosphor. The rates of incorporation of 14C glycine into creatinine over 7 days were slightly increased (for S. W., 0.21% vs 0.23%; for E. S., 0.35% vs. 0.39% of the administered dose of glycine in control and fructose studies, respectively) by fructose infusion; both values fell within the previously described range (0.19x-0.64%).” Patient H. B., who received the largest total dose of fructose but in whom the initial rate of infusion was lowest, showed by far the smallest increment in corrected incorporation of 14C glycine into urinary uric acid (Table 2). This patient also showed a marked increase in urate pool size but a decrease in urate turnover and uncorrected 14Cglycine incorporation, as well as a minimal increment in urinary uric acid excretion on the day of infusion and an overall decrease in urinary uric acid excretion. These findings, in addition to the unexplained decrement in cumulative urinary 15N recovery following infusion and the rather large discrepancy between values for turnover excreted and “N recovery, are consistent with minimal increase in purine synthesis de novo as a result of the altered schedule of fructose infusion in this patient. Specific radioactivities of urinary uric acid with time in control studies showed variation in the magnitude and time of appearance of peak specific activities similar to those previously reported for both normal and gouty individuals.” After fructose administration, the magnitude of the peak specific activities increased in all cases but patient H. B., and the peak specific activity appeared during day 2 for patients R. M., T. B., and H. B., and day 3 for
PURINE
867
SYNTHESIS
0
I
2
3
5
4
TIME
6
7
6
9
IO
(days)
Fig. 2. Specific activities of urinary uric acid in two patients after control and fructose-infusion studies in which oral “C glycine was administered. Values shown for specificactivities in fructoseinfusion studies have been corrected as previously described. A - - - - A, R. M. control study; l - - - l, R. M., fructose study; a - - - - a, E. S. control study; o - --o, E. 5. fructose study.
patients E. S. and S. W. The specific activities trol and fructose studies are shown for patients
of urinary uric acid during R. M. and E. S. in Fig. 2.
con-
DISCUSSION
After fructose administration, the increase in the pool size of urate (Table 2). as well as the increases in plasma urate and in urinary excretion of urate, indicate that uric acid production is rapidly enhanced. Possible mechanisms involved are: (A) increased breakdown of preformed purine nucleotides and derivatives, (B) increased synthesis of purine nucleotides de novo, and (C) a combination of (A) and (B). Studies on liver-adenine nucleotide concentrations in the rat have clearly shown that the first of these mechanisms is operative after fructose administration.4-8 In addition, however, our findings imply that the second mechanism operates as well and extend to intravenous fructose infusion the recent observation by Emmerson’ of increased 14C glycine incorporation into urinary uric acid resulting from oral fructose administration. That the increased glycine incorporation reflects increased purine synthesis de novo rather than depletion of the glycine pool resulting from fructose loading is indicated by our demonstration in two patients that the rates of 14C glycine incorporation into urinary creatinine following fructose infusion are increased only lo:/; beyond control rates despite two-fold increases in the rates of incorporation of the isotope into urinary uric acid. We are unable to ascertain from our studies whether the increase in the rate of purine synthesis is a primary effect of a metabolite of fructose or is secondary
868
RAM0
ET Al.
to the very transient nucleotide depletion’ with concomitant release of feedback inhibition.19 Oral administration of 14C glycine can be regarded as a “pulse labeling” of the glycine pool with the isotope. Since turnover of the glycine pool is rapid, analysis of the patterns of labeling of urinary uric acid over the course of days following fructose infusion is too insensitive a method to distinguish between these mechanisms of alteration of purine synthesis. Consideration of a potential mechanism whereby infusion of fructose could result in primary stimulation of purine synthesis de novo directs attention to the concentration of PP-ribose-P which appears to be an important regulator of the rate of purine-nucleotide synthesis.‘9*20Among the metabolic alterations in liver tissue caused by fructose are increases in the concentrations of triose phosphates and fructose-6-phosphate,’ both substrates of the nonoxidative pentose-phosphate pathway. Simultaneous increases in hepatic concentrations of the pentose phosphates and PP-ribose-P, however, remain to be demonstrated, and the results of our measurements, (as well as those of Fox and Kelley2) of erythrocyte PP-ribose-P concentrations during fructose infusion fail to confirm such an increase in this cell. In addition, since PP-ribose-P synthesis is dependent on the inorganic phosphate concentration,” diminution of the hepatic intracellular inorganic phosphate concentration incident to fructose infusion6v7 could limit the production of PP-ribose-P. For these reasons, an increase of intracellular PP-ribose-P concentration as the basis of fructoseinduced increased purine synthesis de novo remains a hypothesis as unsubstantiated as a similar suggestion concerning the mechanism of increased purine synthesis in Type 1 glycogen-storage disease.22 Patient H. B., who received the largest total dose of fructose but without the very rapid early phase of infusion (Table I), showed the smallest increment in 14Cglycine incorporation. While this finding might be explained in terms of the rapidity of turnover of the glycine pool prior to delivery of an adequate dose of fructose, we interpret the decrease in urate turnover and in uncorrected glycine incorporation in this patient as indicating a real failure of the high-dose infusion to increase purine synthesis de novo to a degree comparable to those infusions of fructose in which a large early dose was given. These findings are compatible with the previous suggestion 23of a time-dependency as well as dosedependency governing the hyperuricemic response to fructose administration. Patient H. B.‘s increased pool size and mild hyperuricemic response to fructose infusion most likely reflects increased breakdown of preformed nucleotides alone. Since patients T. B. and H. B. share the same genetic abnormality of purine metabolism,” the higher g 1ytine incorporation of the former in response to fructose infusion could well result from the higher rate of infusion. At the lower infusion rate, then, increased uric-acid production may result from breakdown of preformed nucleotides, while an enhancement of purine synthesis de novo is produced at more rapid infusion rates. REFERENCES K: 1. Perheentupa J, Raivio induced hyperuricaemia. Lancet 1967
Fructose2528-53
1,
2. Fox IH, Kelley WN: Studies on the mechanism of fructose-induced hyperuricemia in man. Metabolism 21:713-721, 1972
PURINE
869
SYNTHESIS
3. Simkin PA: Hexose infusion in Cebus monkeys: Effects on uric acid metabolism. Metabolism 21:1029-1036, 1972
4. Maenpail PH, Raivio KO, Kekomlki MP: Liver adenine nucleotides: Fructose-induced depletion and its effect on protein synthesis. Science 161:1253~1254, 1968 5. Raivio KO, Kekomlki MP, Maenpll PH: Depletion of liver adenine nucleotides induced by D-fructose. Dose-dependence and specificity of the fructose effect. Biochem Pharmacol 18: 261552624. 1968 6. Woods The cause of l-phosphate il9:501-510,
HF, Eggleston LV, hepatic. accumulation on fructose loading. 1970
7. Burch HB, Max P Jr, Chyu hydroxyacetone, metabolites and and kidney. J Biol
Krebs HA: of fructose Biochem J
Lowry OH, Meinhardt L, K: Effect of fructose, diglycerol, and glucose on related compounds in liver Chem 245:2092-2102, 1970
8. Bode Ch, Schumacher H, Goebell H, Zelder 0, Pelzel H: Fructose-induced depletion of liver-adenine nucleotides in man. Hormone Metab Res 3:289-290, 1971
13. Taussky HH: A microcolorimetric determination of creatinine in urine by the Jaffe reaction. J Biol Chem 208:853-861, 1954 14. Grayzel Al, Seegmiller JE. Love E: Suppression of uric-acid synthesis in the gouty human by the use of 6-diazo-5-oxo-L-norleucine. J Clin Invest 39:4477454, 1960 15. McCarthy JJ, Eppley RW: A comparison of chemical, isotopic, and enzymatic methods for measuring nitrogen assimilation of marine phytoplankton. Limnol Oceanogr 17:37lL381. 1972 16. Elliott WC, Cohen LS, Klein MD, Lane FJ, Gorlin R: Effects of rapid fructose infusion in man. J Appl Physiol23:865-869, 1967 17. Yii TF. Sirota JH, Berger L, Halpern M. Gutman AB: Effect of sodium lactate on urate clearance in man. Proc Sot Exp Biol Med 96: 809-813, 1957 18. Benedict SR: Studies in creatine and creatinine metabolism. I. The preparation of creatine and creatinine from urine. J Biol Chem 18:183-190, 1914
Al, Laster L, in gout. J Clin
19. Becker MA, Seegmiller JE: Genetic aspects of gout. Ann Rev Med 25: 15-28, 1974 20. Fox IH, Kelley WN: Phosphoribosylpyrophosphate in man: Biochemical and clinical significance. Ann Int Med 74:424-433, 197 I 21. Hershko A, Razin A, Mager J: Regulation of the synthesis of 5-phosphoribosyl-lpyrophosphate in intact red blood cells and in cell-free preparations, Biochim Biophys Acta 184164-76, 1969 22. Alepa FP, Howell RR, Klinenberg JR, Seegmiller JE: Relationships between glycogen
12. Liddle L, Seegmiller JE. Laster L: The enzymatic spectrophotometric method for determination of uric acid. J Lab Clin Med 54: 903-913. 1959
storage disease and tophaceous gout. Am J Med 42:58-66, 1967 23. Heuckenkamp P-U, Zollner N: Fructoseinduced hyperuricaemia. Lancet 1:8088809. 1971
9. Emmerson, urate production. I974
BT: Effect of oral fructose on Ann Rheum Dis 33:276-280,
IO. Becker MA, Meyer LJ, Seegmiller JE: Gout with purine overproduction due to increased phosphoribosylpyrophosphate tase activity. Am J Med 55:232-242, I I. Seegmiller JE, Grayzel Liddle L: Uric-acid production lnvest40:1304-1314. 1961
synthe1973
of Human Purine Synthesis by Fructose Infusion
Kari 0. Raivio, Michael A. Becker, Laurence J. Meyer, Martin George
Nuki, and J. Edwin
In order to clarify the mechanism of hyperuricemia and hyperuricosuria resulting from rapid infusion of fructose in man, the effects of an intravenous infusion of 125200 g of fructose given over 3-4 hr on the rate of purine synthesis de novo was measured in one individual with osteoarthritis and four patients with gout. The incorporation of l-“C glycine into urinary uric acid was measured, and the pool size and turnover of urate were assessed by renal excretion of simultaneously administered 15N urate. Fructose caused an expansion of body urate pool in all subjects, while urate turnover was increased in four. The rate of incorporation of “C glycine into urinary uric acid corrected for extrarenal disposal was increased in all cases (21%430%). In two patients, rates of incorporation of “C glycine into urinary creatinine were increased by 10% and 1 1 %, while
De Novo L. Greene,
Seegmiller
rates of incorporation into uric acid were increased 84% and 159%, respectively, as a result of fructose infusion. Specific enhancement of the rate of purine synthesis de novo was suggested by these findings. The rate of infusion appeared more important than total dose in determining the magnitude of this effect. Whether the increased rate of purine synthesis was a result of direct stimulation by a fructose metabolite or was secondary to fructoseinduced purine nucleotide depletion is uncertain, since the kinetics of glycine incorporation were consistent with either mechanism. Erythrocyte PP-ribose-P concentrations, however, were diminished during infusion rather than increased as might be expected if fructose infusion stimulated purine synthesis by increasing availability of this regulatory substrate.
I
NTRAVENOUS ADMINISTRATION of D-fructose produces increases in serum urate concentration and in urinary uric acid excretion in several mammalian species.lm3 A rapid breakdown of liver adenine nucleotides related to the rapid phosphorylation of fructose has been postulated to account for these effects,4m6 and decreases in liver adenine nucleotides have been measured during fructose loading the rat4,6v7and in man.* In a recent report, EmmersonY has assessed the effects of orally-administered fructose on the rate of incorporation of 14C glycine into urinary uric acid in man and has suggested an increased rate of purine synthesis de novo during fructose feeding. The purpose of the present study was to investigate whether the hyperuricemia and hyperuricosuria associated with intravenous infusion of fructose involves an increased rate of
From rhe Division of Rheumatology, Department of Medicine. University oj’ Califbrnia at San Diego and San Diego Velerans Administration Hospital. La Jolla, Calif: Receivedforpublication November8. 1974. Supported in part bv grants A M-13622, AM-05646, and MG-I 7702 of the National Instirures oJ Health and grants from the United States Veterans Administration. the Arthritis Foundation. the National Generics Foundation, and the National Foundation. Dr. Ravio was a fellow of the Arthritis Foundation and Dr. Nuki was a Merck Fellow in Clinical Pharmacology. Reprint requests should be addressed to Dr. J. Edwin Segmiller. Department of Medicine, M-013, University of Calvbrnia. San Diego, La Jolla, Cali/: 92037 .=I1975 bv Grune & Stratton. Inc.
Meiobolism,Vol.
24, No. 7(July), 1975
661
49
15
51
43
E. S.
SW.
T. B.
H. B.
values
57
KM.
Normal
w
Subject
Age
normal
excessive
of hip
increased synthetase
PP-ribose-P activity 10
uric acid production;
excessive
activity”
Gout;
PP-ribose-P
increased
synthetase
production;
excessive
uric-acid
Gout,
production
uric-acid
Gout,
production
uric-acid
Gout;
Osteoarthritis
Diagnosis
4
7
7
10
4
(days)
of study
Duration
210
510
510
580
550
mg/kg
10 min.
Initial
2630
1405
1405
1640
1560
Total
Dose of fructose
9.9
9.4
9.2
7.8
4.2
mg%
3.0-7.0
Baseline
Plasma
Urote
11.5
12.0
11.0
9.0
5.1
Infusion
During
413
980
1405
650
490
470
f
75 SD”
1019
1616
032
008
507
hr
Infusion
Hours of
First 24
Uric Acid
mgf24
Baseline
Urinarv
1650
15-25
1550
1725
1660
2060
mg/kg
1670
24 hr
1606
1608
1680
2140
hr
Infusion
Hours of
First 24
Creatininc
mg/24
Baseline
Urinary
Table 1. Clinical Summary of Study Patients and Effects of Fructose Infusion on Measurements of Purine Metabolism
2.8
5.0
5.1
3.0
3.2
2.7
f
nmoles/ml
Baseline
Eryihrocyie
0.5
SD”
4.5
4.0
2.8
2.8
2.2
pocked cells
Infusion
PP-ribose-P
PURINE
purine effect.
863
SYNTHESIS
nucleotide
synthesis
and, if possible,
MATERIALS
to define the mechanism
of such an
AND METHODS
Five male subjects were studied after each was given a full explanation of the implications of the studies and informed consent was obtained. Clinical characteristics of these individuals are summarized in Table 1. Renal function, as measured by creatinine clearance, was normal in all patients as was erythrocyte hypoxanthine-guanine-phosphoribosyltransferase activity.” Two patients had excessive activity of PP-ribose-P synthetase to account for their purine overproduction.” All were hospitalized on a metabolic ward and received no radiographic contrast materials or medications other than colchicine during the course of the studies. After a minimum of 5 days on a 2600 cal purine-free diet with I g protein/kg body weight, a control isotope study was carried out, followed at an interval of IO-14 days by a second isotope study in association with an infusion of fructose. The rate of purine synthesis de novo was estimated in each individual by measurement of the incorporation of 1-14C glycine (SrCi, 18.75 mCi/mmole, New England Nuclear Corporation) into urinary uric acid for periods ranging from 4 to IO days after oral administration of were corrected for extrathe isotope with breakfast milk. Values for 14C glycine incorporation renal disposal of urate as determined by simultaneous intravenous injection of lithium 1,3-“N urate (20 mg, 46.3 atom % excess, Isomet Corporation) in SY:, glucose.” Urine was collected in two l2-hr samples during the first day of each study and in 24-hr samples thereafter and was stored at room temperature with 3 ml of toluene as preservative. After analysis of urine samples for uric acidI and creatinine13 concentrations, uric acid was isolated and its content of 14C I4 and 15N Is was determined. Body urate pool and turnover, as well as corrected cumulative incorporation of label from glycine, were calculated as previously described.” On the morning of fructose infusion, oral intake of 250 ml of water every 30 min was begun I hr prior to infusion and continued during its course. Intravenous infusion of fructose was begun at the time of oral isotope administration. D-fructose, loo/, solution in water (Abbott Laboratories, Chicago), was administered as a rapid infusion of 450 ml over IO min followed by a 4-hr infusion at the rate of 200 ml/hr. Patient H.B. received a higher total dose of fructose but at a less-rapid rate during the initial IO min of infusion: 500 ml of the fructose solution over 30 min, followed by 500 ml over the next 60 min, and 1000 ml over the following 90 min. In all cases, except patient H.B., the dose of fructose during the initial 10 min of infusion exceeded 0.5 g/kg (Table I). a dose consistently associated epigastric discomfort during
with a hyperuricemic effect2 All subjects complained of transient the rapid injection of fructose, but no alarming objective signs de-
veloped. This symptom has previously been observed in conjunction with fructose administration.” Patients were allowed their regularly scheduled purine-free breakfasts during the infusion. In order to correct the isotopic data obtained in the fructose-infusion experiments for residual ‘sotope enrichment from the preceding control studies, the specific radioactivity of the urinary uric acid isolated throughout the control study was plotted as a function of time, and the values obtained by extrapolating the curve were subtracted from the 14C enrichments determined in the subsequent study. The validity and reproducibility of this method was studied by performance of two successive control studies at an interval of IO days in another individual. Using the method of correction described here, agreement to within 1% was found in this patient between the two values of corrected 14C glycine incorporation into urinary uric acid over 7 days. During the course of fructose infusion, blood samples for assay of plasma uric acid” and erythrocyte 5-phosphoribosyl I-pyrophosphate (PP-ribose-P)” concentrations were obtained at 0, IO, 30, 45, 60, 120. 180, and 240 min.
RESULTS
In agreement with earlier studies,” all subjects showed an increase (mean 22:,;,, range 167~;~28%) in plasma uric acid concentration with a peak at 30-120 min after the start of the fructose infusion (Table 1). Urinary uric acid excre-
1202
2321
2502
5. w. Control
Fructose
Fructose
H. B. Control
Fructose
T. 8. Control
0.48
1.34
0.56
1317
2815
0.72
0.65
2412
3585
0.46
0.47
0.54
1164
2518
Fructose
0.60
0.89
E. s. Control
2509
Fructose
Subject
R.M. Control
Turnover Rate K pools/day
1576
1770
2337
1736
1150
1100
1200
628
1505
1069
TUVlOVl?~ KA mg/day
Effects of Fructose infusion on Incorpomtion
Urote Pool Sire A mg
Table 2.
867
980
1439
1405
690
650
550
490
510
470
Average Daily Uric Acid Excretion E mg/day
of Isotopically
labeled
55
55
62
a0
60
59
46
78
35
44
TWnOVW Excreted E/KA %
40
57
60
78
47
50
51
62
41
44
Uric Acid
0.42
0.48
3.90
1.17
0.41
0.24
0.65
0.30
0.25
0.08
1.02
0.84
6.50
1.50
0.88
0.48
1.27
0.49
0.62
0.18
Administered Glycine Uncorrected Corrected G G/U % %
Cumulative Recovery of Isotope in Urinary Uric Acid
into Urinary
Administered Uric Acid U %
Uric Acid and Glycine
E
PURINE
865
SYNTHESIS
tion during the first 24 hr after beginning infusion was also increased; the range of this increase was 4%-65x above control values for excretion, and the mean increase was 27%. In contrast, a simultaneous decrease ranging between 87; and 21% with a mean of 14% was observed for erythrocyte PP-ribose-P concentration in all patients. The magnitude of this decrease was greatest between 30 and 90 min of infusion and was sustained through the remainder of the infusion. The changes in plasma urate and erythrocyte PP-ribose-P concentrations were transient and returned to baseline within 24 hr, while urinary uric acid excretion remained increased up to 48 hr. The results of the isotope studies are summarized in Table 2. Fructose infusion might be expected to disturb the steady state of urate pool size and turnover rate and thus render inaccurate the interpretation of measurements of these based on isotopic uric-acid injection. However, for each patient, semilog plot of uric acid isotope concentration versus time yielded a linear relationship as exemplified for patient T. B. in Fig. 1. Correlation coefficients for the least squares fit line relating log of uric-acid isotopic concentration to time varied from 0.92 to 0.99 among the five patients. Extrapolation of this line to time 0 was therefore used to estimate pool size, and turnover rate was estimated from
-2
78 SLOPE=-0652 DAYS-’ IA’JERCEPJ= -I 35 = ln 0 259
-3
In1
0
I
2
3 TIME
4
5
6
7
(days)
Fig. 1. Decline in isotope content ( 1) of urinary uric acid in patient T. B. ofter fructose infusion. Pool size was estimated from extrapolation of experimental line to time 0, and turnover rate was estimated from the slope of the line.
866
RAM0
ET Al.
the slope of the line. In each patient, fructose caused an increase in the urate pool which more than doubled in three cases. The turnover rate of the urate pool decreased in four of the subjects, raising the possibility that fructoseinduced lacticacidemia was impairing renal secretion of uric acid as previously described.” However, in agreement with a previous study,’ no consistent decrease in uric-acid clearance (during or after infusion) could be demonstrated, and turnover of urate was actually increased in all four subjects given an initial rapid rate of infusion. In addition, the cumulative recovery of administered “N urate in the urine was decreased by as much as 30% following fructose infusion, an observation difficult to explain on the basis of the transient lactic acidosis resulting from acute fructose loading. (In patient T. B., serial measurements of blood lactate during infusion showed a four-fold increase in lactate concentration to 33 mg/lOO ml at 90 min with a return to baseline value by 240 min.) Cumulative incorporation of 14Cglycine into urinary uric acid was markedly increased (range: 21%-430x) by fructose infusion in all cases, suggesting an enhanced rate of purine synthesis de novo (Table 2). (The magnitude of increase in incorporation was relatively little influenced by the length of the study, from 4 to 10 days.) An alternative explanation for the increased incorporation of 14C glycine into urinary uric acid is that fructose infusion diminished the endogenous glycine pools of tissues responsible for purine-nucleotide synthesis. This possibility was investigated by comparing cumulative 14Cglycine incorporation into urinary creatinine during the control and fructose studies of two patients (S. W. and E. S.). Creatinine was purified by the Benedict method” and isotopic enrichment was measured by liquid scintillation counting at 77% efficiency in Aquasol phosphor. The rates of incorporation of 14C glycine into creatinine over 7 days were slightly increased (for S. W., 0.21% vs 0.23%; for E. S., 0.35% vs. 0.39% of the administered dose of glycine in control and fructose studies, respectively) by fructose infusion; both values fell within the previously described range (0.19x-0.64%).” Patient H. B., who received the largest total dose of fructose but in whom the initial rate of infusion was lowest, showed by far the smallest increment in corrected incorporation of 14C glycine into urinary uric acid (Table 2). This patient also showed a marked increase in urate pool size but a decrease in urate turnover and uncorrected 14Cglycine incorporation, as well as a minimal increment in urinary uric acid excretion on the day of infusion and an overall decrease in urinary uric acid excretion. These findings, in addition to the unexplained decrement in cumulative urinary 15N recovery following infusion and the rather large discrepancy between values for turnover excreted and “N recovery, are consistent with minimal increase in purine synthesis de novo as a result of the altered schedule of fructose infusion in this patient. Specific radioactivities of urinary uric acid with time in control studies showed variation in the magnitude and time of appearance of peak specific activities similar to those previously reported for both normal and gouty individuals.” After fructose administration, the magnitude of the peak specific activities increased in all cases but patient H. B., and the peak specific activity appeared during day 2 for patients R. M., T. B., and H. B., and day 3 for
PURINE
867
SYNTHESIS
0
I
2
3
5
4
TIME
6
7
6
9
IO
(days)
Fig. 2. Specific activities of urinary uric acid in two patients after control and fructose-infusion studies in which oral “C glycine was administered. Values shown for specificactivities in fructoseinfusion studies have been corrected as previously described. A - - - - A, R. M. control study; l - - - l, R. M., fructose study; a - - - - a, E. S. control study; o - --o, E. 5. fructose study.
patients E. S. and S. W. The specific activities trol and fructose studies are shown for patients
of urinary uric acid during R. M. and E. S. in Fig. 2.
con-
DISCUSSION
After fructose administration, the increase in the pool size of urate (Table 2). as well as the increases in plasma urate and in urinary excretion of urate, indicate that uric acid production is rapidly enhanced. Possible mechanisms involved are: (A) increased breakdown of preformed purine nucleotides and derivatives, (B) increased synthesis of purine nucleotides de novo, and (C) a combination of (A) and (B). Studies on liver-adenine nucleotide concentrations in the rat have clearly shown that the first of these mechanisms is operative after fructose administration.4-8 In addition, however, our findings imply that the second mechanism operates as well and extend to intravenous fructose infusion the recent observation by Emmerson’ of increased 14C glycine incorporation into urinary uric acid resulting from oral fructose administration. That the increased glycine incorporation reflects increased purine synthesis de novo rather than depletion of the glycine pool resulting from fructose loading is indicated by our demonstration in two patients that the rates of 14C glycine incorporation into urinary creatinine following fructose infusion are increased only lo:/; beyond control rates despite two-fold increases in the rates of incorporation of the isotope into urinary uric acid. We are unable to ascertain from our studies whether the increase in the rate of purine synthesis is a primary effect of a metabolite of fructose or is secondary
868
RAM0
ET Al.
to the very transient nucleotide depletion’ with concomitant release of feedback inhibition.19 Oral administration of 14C glycine can be regarded as a “pulse labeling” of the glycine pool with the isotope. Since turnover of the glycine pool is rapid, analysis of the patterns of labeling of urinary uric acid over the course of days following fructose infusion is too insensitive a method to distinguish between these mechanisms of alteration of purine synthesis. Consideration of a potential mechanism whereby infusion of fructose could result in primary stimulation of purine synthesis de novo directs attention to the concentration of PP-ribose-P which appears to be an important regulator of the rate of purine-nucleotide synthesis.‘9*20Among the metabolic alterations in liver tissue caused by fructose are increases in the concentrations of triose phosphates and fructose-6-phosphate,’ both substrates of the nonoxidative pentose-phosphate pathway. Simultaneous increases in hepatic concentrations of the pentose phosphates and PP-ribose-P, however, remain to be demonstrated, and the results of our measurements, (as well as those of Fox and Kelley2) of erythrocyte PP-ribose-P concentrations during fructose infusion fail to confirm such an increase in this cell. In addition, since PP-ribose-P synthesis is dependent on the inorganic phosphate concentration,” diminution of the hepatic intracellular inorganic phosphate concentration incident to fructose infusion6v7 could limit the production of PP-ribose-P. For these reasons, an increase of intracellular PP-ribose-P concentration as the basis of fructoseinduced increased purine synthesis de novo remains a hypothesis as unsubstantiated as a similar suggestion concerning the mechanism of increased purine synthesis in Type 1 glycogen-storage disease.22 Patient H. B., who received the largest total dose of fructose but without the very rapid early phase of infusion (Table I), showed the smallest increment in 14Cglycine incorporation. While this finding might be explained in terms of the rapidity of turnover of the glycine pool prior to delivery of an adequate dose of fructose, we interpret the decrease in urate turnover and in uncorrected glycine incorporation in this patient as indicating a real failure of the high-dose infusion to increase purine synthesis de novo to a degree comparable to those infusions of fructose in which a large early dose was given. These findings are compatible with the previous suggestion 23of a time-dependency as well as dosedependency governing the hyperuricemic response to fructose administration. Patient H. B.‘s increased pool size and mild hyperuricemic response to fructose infusion most likely reflects increased breakdown of preformed nucleotides alone. Since patients T. B. and H. B. share the same genetic abnormality of purine metabolism,” the higher g 1ytine incorporation of the former in response to fructose infusion could well result from the higher rate of infusion. At the lower infusion rate, then, increased uric-acid production may result from breakdown of preformed nucleotides, while an enhancement of purine synthesis de novo is produced at more rapid infusion rates. REFERENCES K: 1. Perheentupa J, Raivio induced hyperuricaemia. Lancet 1967
Fructose2528-53
1,
2. Fox IH, Kelley WN: Studies on the mechanism of fructose-induced hyperuricemia in man. Metabolism 21:713-721, 1972
PURINE
869
SYNTHESIS
3. Simkin PA: Hexose infusion in Cebus monkeys: Effects on uric acid metabolism. Metabolism 21:1029-1036, 1972
4. Maenpail PH, Raivio KO, Kekomlki MP: Liver adenine nucleotides: Fructose-induced depletion and its effect on protein synthesis. Science 161:1253~1254, 1968 5. Raivio KO, Kekomlki MP, Maenpll PH: Depletion of liver adenine nucleotides induced by D-fructose. Dose-dependence and specificity of the fructose effect. Biochem Pharmacol 18: 261552624. 1968 6. Woods The cause of l-phosphate il9:501-510,
HF, Eggleston LV, hepatic. accumulation on fructose loading. 1970
7. Burch HB, Max P Jr, Chyu hydroxyacetone, metabolites and and kidney. J Biol
Krebs HA: of fructose Biochem J
Lowry OH, Meinhardt L, K: Effect of fructose, diglycerol, and glucose on related compounds in liver Chem 245:2092-2102, 1970
8. Bode Ch, Schumacher H, Goebell H, Zelder 0, Pelzel H: Fructose-induced depletion of liver-adenine nucleotides in man. Hormone Metab Res 3:289-290, 1971
13. Taussky HH: A microcolorimetric determination of creatinine in urine by the Jaffe reaction. J Biol Chem 208:853-861, 1954 14. Grayzel Al, Seegmiller JE. Love E: Suppression of uric-acid synthesis in the gouty human by the use of 6-diazo-5-oxo-L-norleucine. J Clin Invest 39:4477454, 1960 15. McCarthy JJ, Eppley RW: A comparison of chemical, isotopic, and enzymatic methods for measuring nitrogen assimilation of marine phytoplankton. Limnol Oceanogr 17:37lL381. 1972 16. Elliott WC, Cohen LS, Klein MD, Lane FJ, Gorlin R: Effects of rapid fructose infusion in man. J Appl Physiol23:865-869, 1967 17. Yii TF. Sirota JH, Berger L, Halpern M. Gutman AB: Effect of sodium lactate on urate clearance in man. Proc Sot Exp Biol Med 96: 809-813, 1957 18. Benedict SR: Studies in creatine and creatinine metabolism. I. The preparation of creatine and creatinine from urine. J Biol Chem 18:183-190, 1914
Al, Laster L, in gout. J Clin
19. Becker MA, Seegmiller JE: Genetic aspects of gout. Ann Rev Med 25: 15-28, 1974 20. Fox IH, Kelley WN: Phosphoribosylpyrophosphate in man: Biochemical and clinical significance. Ann Int Med 74:424-433, 197 I 21. Hershko A, Razin A, Mager J: Regulation of the synthesis of 5-phosphoribosyl-lpyrophosphate in intact red blood cells and in cell-free preparations, Biochim Biophys Acta 184164-76, 1969 22. Alepa FP, Howell RR, Klinenberg JR, Seegmiller JE: Relationships between glycogen
12. Liddle L, Seegmiller JE. Laster L: The enzymatic spectrophotometric method for determination of uric acid. J Lab Clin Med 54: 903-913. 1959
storage disease and tophaceous gout. Am J Med 42:58-66, 1967 23. Heuckenkamp P-U, Zollner N: Fructoseinduced hyperuricaemia. Lancet 1:8088809. 1971
9. Emmerson, urate production. I974
BT: Effect of oral fructose on Ann Rheum Dis 33:276-280,
IO. Becker MA, Meyer LJ, Seegmiller JE: Gout with purine overproduction due to increased phosphoribosylpyrophosphate tase activity. Am J Med 55:232-242, I I. Seegmiller JE, Grayzel Liddle L: Uric-acid production lnvest40:1304-1314. 1961
synthe1973
