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Studies on the mechanism of fructose-induced hyperuricemia in man
Studies on the Mechanism of Fructose-induced Hyperuricemia in Man By Irving
t-i. Fox and William
The rapid intravenous infusion of fructose produces a transient increase in plasma urate concentration, as well as an increase in the urinary excretion of oxypurines and uric acid. Fructoseinduced hyperuricemia and hyperuricaciduria is associated with a striking increase in the blood lactate concentration, a decrease in erythrocyte phosphoribosylpyrophosphate (PP-riboseP) and ribosed-phosphate concentration, and no detectable change in ery-
N. Kelley
throcyte ATP concentration. Although pretreatment with allopurinol prevents the hyperuricemic effect of fructose, the increase in plasma lactate is not modified and the increase in urinary enhanced. oxypurine excretion is These results are consistent with the hypothesis, based on previous studies in the rat, that fructose-induced hyperuricemia in man results from an increased degradation of purine ribonucleotides.
H
clinical disorder. However, in many YPERURICEMIA is a common instances the precise mechanisms responsible for the elevated serum urate concentration are not well understood. Elucidation of the mechanism by which certain normal metabolic intermediates and their structural analogues alter the serum urate concentration may be useful in delineating potential pathophysiologic alterations leading to hyperuricemia. This approach proved to be valuable in defining the deficiency of hypoxanthine-guanine phosphoribosyl transferase in the Lesch-Nyhan syndrome and in some patients with gout.’ The infusion of large doses of fructose in man initiates a number of biochemical changes.“-‘” Many of these alterations, such as the development of hyperlactacidemia, the decrease in serum inorganic phosphate, and the decrease in plasma glucose can be attributed to the phosphorylation of fructose and the entrance of this phosphorylated intermediate into the glycolytic pathway. A striking increase in the serum urate concentration has also been observed following the infusion of fructose.4’g However, the mechanism responsible for this effect has been unclear. In the present study, we have confirmed the observation that fructose produces hyperuricemia and have examined the mechanism. Our data in man provide support for previous conclusions based on studies in the rat in which hyperuricemia was attributed to an accelerated catabolism of purine nucleotides.11-13 From fhe Division of Rheumntic and Genetic Diseases, Depnrfment of Medicine, Drrke University Medical Center, Durham, N. C. Received for publication January 7, 1972. Supported by USPHS Grant AM 14362. Irving H. Fox, M.D.: Postdoctornl Fellow of the Medical Research Council of Canada; Research Fellow, Division of Rheumntic and Genetic Diseases, Duke Uniuersify Medical Center, Durham, N. C. William N. Kelley, M.D.: Chief, Division of Rheumntic and Genetic Diseases, and Associate Professor of Medicine, Assistant Professor of Biochemistry, Duke University Medical Center, Durhnm, N. C. Metabolism,
Vol. 21, No. 8 (August), 1972
713
Range
M
2 1 S.D.3*
t Mean
1 S.D.3l
k
* Mean
(D.H. F7 2 420)
M
(D.H. JO1630) L.L.
M
M
J.W.
(D.H. H93284)
(D.H. J13731) H.M.
Patients A.B.
Normal
Sex
61
60
48
75
Age (Yr)
a.a+o.l
8.5k0.2
5.7kO.l
5420.4
3-7
Plasma Urate (mg/lOO ml)
ia
31
16
44
6-25
UV,,ste (mg/hr)
1. Clinical
-
33
12
35
106
145
86
142
(@moles/ C,, (ml/min) hr)
osspurines
uv
Table
5.5
5.9
4.7
13.6
C,,,t, (ml/min)
5.2
4.1
5.5
9.6
5.412.1+
C,,,,t,/C,, ( x 100)
Summary
-
0.77
-
0.86
ATP (mW .w
5.7k1.4
4.o-co.7
3.7kO.9
2.3-cO.3’
4.4&l
(,llW
-
1.4-co.4
1.420.4
-
Ribose-5-P CPM)
Concentration
PP-ribose-P
Erythrocyte
127
108
100
110
98ei4t
mg/hr)
(nmolesl
Hypoxanthine
13
29
40
31
31 i61
mg/hr)
Adenine (nmoles/
Erythrocyte Lysate Phosphoribosyltransferase Activity
STUDIES
OF FRUCTOSE-INDUCED
715
HYPERURICEMIA
MATERIALS
AND METHODS
Four male patients with gout were admitted to the Clinical Research Unit at Duke University Medical Center. The diagnosis of gout was based on the clinical history in all four subjects, the presence of tophi in patients A.B. and J.W., and the demonstration of intracelhrlar monosodium urate crystals by compensated, polarized light microscopy in synovial fluid obtained from subjects L.L. and H.M. during an acute episode of monoarticular arthritis. The patients had essentially normal renal function as measured by glomerular filtration rate. All patients had normal hypoxanthine-guanine phosphoribosyl transferase activity, and one patient was heterozygous for a deficiency of adenine phosphoribosyl transferase (Table 1). Based on the daily excretion of uric acid in the urine after equilibration on a diet essentially free of purines, patient J.W. (654 mg/24 hr) would be classed as an overexcretor, whereas H.M. (394 mg/24 hr), A.B. (441 mg/24 hr), and L.L. (424 mg/24 hr) would be considered normal excretors. The patients received a diet essentially free of purines, with 2200-2600 kcal and 70 g protein for 5 days prior to and throughout the study period. Control studies were performed at least IO days after discontinuing all drugs known to affect uric acid metabolism or excretion. All patients received colchicine, and one patient (A.B.) received quinidine throughout the study period. Studies during allopurinol therapy were performed after the administration of 600 mg/day for at least 48 hr. The patients were fasting, except for water, for 12 hr prior to each study. On the morning of the study, an infusion of isotonic saline was started. Three blood samples were obtained over a I-hr control period. Fructose (0.5 g/kg), sterilized by ultrafiltration, was infused as a 20% solution over a IO-min period. Blood samples were obtained every 15 min during the first hour after the start of the infusion, every 30 min during the second hour, and at the end of the third hour. Urine was collected by voluntary voiding at specified times. Four I-hr collection periods were utilized corresponding to the control period and the first, second, and third hours after the infusion. All blood samples were placed immediately in ice and centrifuged within 15 min at 4°C. Five milliliters of blood, collected in 0.1 ml of 0.2 M EDTA, was processed as previously described, and the extracts assayed for phosphoribosylpyrophosphate (PI’-ribose-I’)14 and ribose-5-phosphate.15 Heparinized blood for ATP assay was extracted using the perchloric acid method of Wailer et al.18 ATP was estimated by the firefly bioluminescence assay.17 Oxalated venous blood for lactate was extracted immediately at 4’C with an equal volume of 7% perchloric acid and assayed by the lactate dehydrogenase assay.18 Uric acid was measured by the uricase method19 and oxypurines were measured by the enzymatic conversion to uric acid.20 Urinary creatinine was determined by the method of Taussky.21 Total reducing sugar was measured on a SMA 6/60, while plasma creatinine and phosphorus were measured on a SMA 12160 using standard techniques. Plasma glucose was measured using the glucose oxidase method. Hypoxanthine-guanine phosphoribosy1 transferase and adenine phosphoribosyl transferase were assayed by radiochemical methods.22 Intact erythrocytes from two gouty patients, including one of the patients (L.L.) who was studied in vivo and one patient with the Lesch-Nyhan syndrome and a deficiency of hypoxanthine-guanine phosphoribosyl transferase (W.E.), were incubated for 2 hr in phosphate-buffered saline as previously described14 without glucose or fructose, with 0.13 M glucose, or with 0.13 M fructose. The erythrocytes were extracted and assayed for PI’-ribose-B.14 When statistical
possible, results significance.
were
analyzed
by
the
paired
two-tailed
Student’s
t test
of
RESULTS The in Table
baseline
data
I. The values
collected presented
in the
four
patients
were obtained
studied
over a I-hr
are
summarized
period
immediately
prior to the fructose infusion and represent the control values for Figs. I and 2. The values for plasma urate, erythrocyte PP-ribose-P, and erythrocyte
FOX AND KELLEY
716
+60r _
-100 +800
c
p\ Fig. 1. Effect of fructose infusion on plasma w-ate and urinary excretion of
uric acid and oxypurines. Results are expressed as per cent of control values L
180
TimelMlnutes)
(Table 1). Solid lines indicate no allopurinol; broken lines indicate pretreatment with allopurinol. Open squares, patient J.W.; black squares, patient L.L.; open circles, patient KM.; and black circles, patient A.B.
ribose-S-phosphate represent the mean obtained during the I-hr control period.
The infusion urate in all four after beginning two patients in therapy. Urinary
*
SD
of fructose produced a significant patients (Fig. IA). The maximum the infusion. There was no rise whom the studies were repeated
Oxypwines
and
of three
different
samples
rise ( p
Uric Acid
As illustrated in Fig. 1B, the infusion of fructose produced a substantial increase in the excretion rate of uric acid. In each patient, the maximal effect occurred in the first hour following the infusion. A modest increase in uric acid excretion was also noted when the fructose was administered to the two subjects receiving allopurinol. In each patient studied, the infusion of fructose also produced a striking increase in the excretion rate of oxypurines (Fig. 1C). The greatest changes were observed during the first hour after the infusion, with levels tapering rapidly back to the control range within 3 hr. The increase in urinary oxypurines was somewhat greater in the two subjects being treated with allopurinol prior to the fructose infusion than that observed in these same subjects before treatment with allopurinol was instituted.
STUDIES
OF FRUCTOSE-INDUCED
717
HYPERURICEMIA
The urine from two of the patients treated with fructose was subjected to anaIysis on an ultraviolet analyzer. The marked rise in urinary oxypurines following intravenous fructose infusion resulted primarily from a rise in hypoxanthine excretion. In addition, a striking increase in the excretion of inosine was also noted. These observations are consistent with the hypothesis that fructose-induced hyperuricemia results from a stimulation of nucleotide catabolism. There was no consistent change in the fractional clearance of uric acid either before or after the treatment with allopurinol (Table 2). Erythrocyte nnd ATP
Phosphovibosylpyrophosphate
(PP-ribose-P),
Ribose-5-phosphate,
As illustrated in Table 3, fructose as well as glucose is capable of stimulating PP-ribose-P production in washed erythrocytes in viva. The infusion of fructose in vivo produced a modest but significant decrease in erythrocyte PP-ribose-P levels in each patient studied (p < 0.05 at 15 and 30 min (Fig. 2). In addition, the intracellular levels of ribose-S-phosphate also began to decrease 45 min after the infusion of fructose in the two patients studied (Fig. 2). The infusion of fructose had no discernible effect on the concentration of ATP in erythrocytes during the 3-hr study period (Fig. 2). Table 2. Effect of Fructose
on Fractional
Excretion
and Clearance
(w/ml)
C “rate&r ( x 100)
0.31 0.40 0.46 0.60 0.17 0.19 0.21 0.18 0.19 0.17 0.15 0.15 0.21 0.48 0.27 0.26 0.29 0.24 0.23 0.25 0.18 0.14 0.16 0.15
9.6 9.1 11.0 14.5 5.2 4.9 5.3 4.8 5.5 4.1 3.7 3.3 4.1 7.3 4.1 4.1 6.1 7.6 7.5 8.0 5.3 4.3 4.8 4.5
U”wate/Ccr Subject
Period
Control 1 2 3 Control 1 2 3 Control 1
A.B.
L.L.
H.M.
2 3 Control 1 2 3 Control 1 2 3 Control 1 2 3
J.W.
A.B.*
J.W.’
l
Receiving
allopurinol,
600 mg/day.
of Uric Acid
718
FOX AND KELLEY
-60 Y E@ t40 2 ,” ;g 0 +E @-40 -80 -%+40 PZF GE” 0 g6 -40 0
30
60
90
120 150 180
Fig. 2. Effect of fructose infusion on concentration of PP-ribose-P, ribose-5phosphate, and ATP in erythrocytes. Results are expressed as per cent of control values (Table 1). Control values represent mean + SD (vertical bars) of three different samples obtained during a 60-min period immediately preceeding infusion of fructose. Open squares, patient J.W.; black squares, patient L.L.; open circles, patient H.M.; and black circles, patient A.B.
Time (Mtnutes)
Table 3. Effect of Glucose and Fructose on Phosphoribosylpyrophosphate Production in Human Erythrocytes In Vitro PP-ribose-P Subject
E.C. W.E. L.L.
Concentration
(pM)
Control
Glucose (0.13 U)
Fructose (0.13 M)
46 201 45
154 358 75
329 455 233
Table 4. Effect of Fructose on Blood Lactate Concentration Blood Lactate Control (mg/lOO ml)
No treatment A.B. H.L.M. J.W. Allopurinol A.B. J.B.
Other The
30 Min After Infusion (mgllO0 ml) (% Change)
6.8 8.9 4.4
28.0 22.0 18.0
+312 +147 +309
6.5 8.0
21.0 30.0
+223 +275
Plnstrm Chemistries infusion
of fructose
crease in the concentration
produced
a transient
of total reducing
the concentration of glucose during the 3-hr study period.
and phosphate
but
substances remained
striking
(fourfold)
in the plasma, essentially
in-
although unchanged
The blood lactate concentration increased markedly 30 min after infusion of fructose (Table 4). This hyperlacticacidemic effect of fructose not altered by pretreatment with allopurinol.
the was
STUDIES
OF FRUCTOSE-INDUCED
HYPERURICEMIA
719
DISCUSSION The phenomenon of fructose-induced hyperuricemia was initially described the hyperuricemic effect of in man by Perheentupa and Raivio. 4 Although fructose has not been consistently confirmed by other investigators,* there appears to be a relationship between the rate of fructose administration and the degree of hyperuricemia produced. g Our studies confirm those of others that fructose, at a dose of 0.5 g/kg infused over a IO-min period, produces a marked and consistent hyperuricemia.
Four possible mechanisms can be formulated to account for this hyperuricemic effect of fructose: (I) a shift in uric acid pool or decreased extrarenal disposal of uric acid leading to an increased filtered load, (2) decreased renal clearance of uric acid, (3) an increased rate of purine synthesis de novo, or (4) an accelerated degradation of purine ribonucleotides. Our data allow the analysis of each of these potential mechanisms. The elevation of urinary oxypurines and the inhibition of fructose-induced hyperuricemia by allopurinol eliminate a shift in the uric acid pool or an alteration of extrarenal disposal of uric acid as an explanation of the abrupt rise in plasma urate. A decreased renal clearance of uric acid would warrant consideration as the mechanism responsible for fructose-induced hyperuricemia, since most drugs that produce hyperuricemia do so by this mechanism.‘” In addition, fructose produces an increase in the plasma lactate concentration,‘-’ and lactic acid itself decreases the renal clearance of uric acid.“” However, the infusion of fructose produced no consistent decrease in the fractional clearance of uric acid. The possibility that fructose may cause hyperuricemia by increasing the rate of purine biosynthesis de novo was considered. As demonstrated in the present study, fructose is both a precursor and stimulator of PI’-ribose-I’ synthesis in human cells in vitro. Since PP-ribose-P is a limiting substrate for the initial step of purine biosynthesis de novo,05’06 an increased concentration of PI’-ribose-P would be expected to increase the rate of purine synthesis de novo. However, several findings indicate that the rapid increase in plasma urate concentration, which occurs 30-45 min after the infusion of fructose, does not appear to be due to this mechanism. (1) The infusion of fructose did not increase PP-ribose-P levels in erythrocytes in vivo; in fact, the intracellular concentration of PP-ribose-P, as well as its immediate precursor, ribose-5-phosphate, appeared to decrease. (2) Others have demonstrated that hyperuricemia occurs following the infusion of fructose to patients with hereditary fructose intolerance in whom there is a genetically determined block in the further metabolism of fructose-l-phosphate.4 This finding also suggests that increased uric acid production can occur without stimulating PI’-ribose-P synthesis. (3) It seems unlikely that an increase in purine biosynthesis de IIOVO would lead to so rapid an increase in the serum urate concentration. The most potent stimulator of purine biosynthesis de novo known in man, 2-ethylamino-1, 3,4-thiadiazole, increases the serum urate concentration in 24-48 hr, not in 30-45 min.37.“s Our observations are most consistant with the hypothesis that fructose
FOX AND KELLEY
720
leads to a rapid degradation of purine nucleotides with the consequent formation of inosine, hypoxanthine, xanthine, and finally uric acid. Recent studies in the rat in vivo, in perfused rat liver, and in human liver have demonstrated that fructose decreases the concentration of adenine nucleotides, especially ATP, and inorganic phosphate within the ce11.11-132”g Since ATP normally inhibits S-nucleotidase and inorganic phosphate inhibits AMP deaminase, these changes would be expected to stimulate the catabolism of AMP to inosine.13 This hypothesis would readilv account for the rapid rise in serum urate concentration and in the urinary excretion of oxypurines and uric acid following the infusion of fructose. The decreased ervthrocyte PPribose-P levels, which occurred prior to the decrease in ribose-s-phosphate concentration, may reflect PP-ribose-P consumption perhaps due to an increased reutilization of hypoxanthine. Only our finding that erythrocyte ATP levels remain unchanged following the infusion of fructose is difficult to reconcile with this hypothesis. However, the concentration of ATP in erythrocytes is normally 400-800 times higher than the concentration of PP-ribose-P and ribose-5-phosphate; it is likely that changes in ATP concentration of the order of magnitude of those observed for PP-ribose-I’ or ribose-5-phosphate would not be detected. Emmerson has recently demonstrated that the chronic oral administration of fructose to three patients led to a small increase in the incorporation of glycine-l-l4 into uric acid in vivo.30 Although this observation is consistent with the hypothesis that fructose increases purine biosynthesis de novo, it is equally consistent with the hypothesis that over a period of days the depletion of purine nucleotides would result in a release of feedback control on purine biosynthesis de novo. ACKNOWLEDGMENT The authors wish to thank Mrs. Dr. J. B. Sidbury, Jr., for measuring McManus for measuring erythrocyte
Margaret Evans for her careful technical assistance, glucose by the glucose oxidase method, and Dr. T. J. ATP concentrations.
REFERENCES 1. Kelley, W. N., Greene, M. L., Rosenbloom, F. M., Henderson, J. F., and Seegmiller, J. E. : Hypoxanthine-guanine phosphoribosyltransferase deficiency in gout. Ann. Intern. Med. 70:155, 1969. 2. Miller, M., Craig, J. W., Drucker, W. R., and Woodward, H.: The metabolism of fructose in man. Yale J. Biol. Med. 29:335, 1956. 3. Froesch, E. R. : Essential fructosuria and hereditary fructose intolerance. In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S. (Eds.): The Metabolic Basis of Inherited Disease. 1966, p. 124. 4. Perheentupa, J., and Raivio, K.: Fructose-induced hyperuricemia. Lancet 2:528, 1967. 5. Bergstrom, J., and Hultman, E.: Syn-
thesis of muscle glycogen in man glucose and fructose infusion. Acta Stand. 182:93, 1967.
after Med.
6. -, -, and Roth-Norlund, A. E.: Lactic acid accumulation in connection with fructose infusion. Acta Med. Stand. 184 :359, 1968. 7. Levin, B., Snodgrass, J. A. I., Oberholzer, V. G., Burgess, E. A., and Dobbs, R.: Fructosaemia, observations of seven cases. Amer. J. Med. 45~826, 1968. 8. Sahebjami, H., and Scalettar, R.: Effects of fructose infusion on lactate and uric acid metabolism. Lancet 1:366, 1971. 9. Heuckenkamp, P-U, and Zollner, N.: Fructose-induced hyperuricaemia. Lancet 1 :808,1971.
STUDIES
OF FRUCTOSE-INDUCED
HYPERURICEMIA
10. Morris, R. C.: An experimental renal acidification defect in patients with hereditary fructose intolerance I. Its resemblance to renal tubular acidosis. J. Clin. Invest. 47:1359, 1968. 11. Maenpaa, I’. H., Raivio, K. O., and Kekomaki, M. I’. : Liver adenine nucleotides: Fructose-induced depletion and its effect on protein synthesis. Science 161:1253, 1968. 12. Raivio, K. O., Kekomaki, M. I’., and Maenpaa, I’. H.: Depletion of liver adenine nucleotides induced by D-fructose. Biochem. Pharmacol. 18:2615, 1968. 13. Woods, H. F., Eggleston, L. V., and Krebs, H. A.: The cause of hepatic accumulation of fructose-l-phosphate on fructose loading. Biochem. J. 119501, 1970. 14. Fox, I. H., Wyngaarden, J. B., and Kelley, W. N.: The depletion of erythrocyte phosphoribosylpyrophosphate by allopurinol in man. New Eng. J. Med. 283:1177,1970. 15. -, and Kelley, W. N.: Human phosphoribosylpyrophosphate synthetase: Kinetic mechanism and end-product inhibition. J. BioI. Chem. 247:2126, 1972. 16. Waller, H., Lohr, G., and Tabatabai, M.: Hamolyse und sehlen zon glucose-6phosphatdehydrogenase n roten blutzellen. Klin. Wschr. 35 :1022, 1957. 17. McManus, T. J., Allen, D. W., and Hyun, D. K.: Unpublished observations. 18. Ohlson, G. F.: Optimal conditions for the enzymatic determination of L-lactic acid. Clin. Chem. 8:1, 1962. 19. Liddle, L., Seegmiller, J. E., and Laster, L.: The enzymatic spectrophotometric method for determination of uric acid. J. Lab. Clin. Med. 54:903, 1959. 20. Klinenberg, J. R., Goldfinger, S., Bradley, K. H., and Seegmiller, J. E.: An enzymatic spectrophotometric method for the determination of xanthine and hypoxanthine. Clin. Chem. 13834, 1967. 21. Taussky, H. H.: A microcolorimetric determination of creatinine in urine by the Jaffe reaction. J. Biol. Chem. 208:853, 1954. 22. Kelley, W. N., Rosenbloom, F. M., Henderson, J. F., and Seegmiller, J. E.: A
721
specific enzyme defect in gout associated with overproduction of uric acid. Proc. Nat. Acad. Sci. USA 57:1735, 1967. 23. Wyngaarden, J. B., and KelIey, W. N.: Gout. The Metabolic In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S. (Eds.): The Metabolic Basis of Inherited Disease (ed. 3). New York, McGraw-Hill, 1972. 24. Howell, R. R., Ashton, D. M., and Wyngaarden, J. B. : Glucose-6-phosphate deficiency glycogen storage disease. Studies on the interrelationships of carbohydrate, lipid and purine abnormalities. Pediatrics 29 :553, 1962. 25. Kelley, W. N., Fox, I. H., and Wyngaarden, J. B.: Essential role of phosphoribosylpyrophosphate in regulation of purine biosynthesis in cultured human fibroblasts. Clin. Res. 18:457, 1970. 26. -, Greene, M. L., Fox, I. H., Rosenbloom, F. M., Levy, R. I., and Seegmiller, J. E.: Effects of erotic acid on purine and lipoprotein metabolism in man. Metabolism 19 :1025, 1970. 27. Krakoff, I. H., and Balis, M. E.: Studies of 2-substituted thiadiazoles in man. J. Clin. Invest. 38 :907, 1959. 25. Seegmiller, J. E., Grayzell, A. I., Liddle, L., and Wyngaarden, J. B.: The effect of ?=ethylamino-1,3,&thiadiazole on the incorporation of glycine into urinary purines and uric acid in man. Metabolism 12:507, 1963. 29. Bode, L., Shumacher, H., Goebell, H., Zelder, O., and Pelzel, H.: Fructose-induced depletion of liver adenine nucleotides in man. Hormone Metab. Res. 3:71, 1971. 30. Emmerson, B.: Personal communication. 31. Smith, M. L., and Scott, J, T.: Uric acid clearance in patients with gout and normal subjects. Ann Rheum. Dis. 30:285, 1970. 32. Fox, I. H., and Kelley, W. N.: Phosphoribosylpyrophosphate in man : Biochemical and clinical significance. Ann. Intern. Med. 74~424, 1971.
t-i. Fox and William
The rapid intravenous infusion of fructose produces a transient increase in plasma urate concentration, as well as an increase in the urinary excretion of oxypurines and uric acid. Fructoseinduced hyperuricemia and hyperuricaciduria is associated with a striking increase in the blood lactate concentration, a decrease in erythrocyte phosphoribosylpyrophosphate (PP-riboseP) and ribosed-phosphate concentration, and no detectable change in ery-
N. Kelley
throcyte ATP concentration. Although pretreatment with allopurinol prevents the hyperuricemic effect of fructose, the increase in plasma lactate is not modified and the increase in urinary enhanced. oxypurine excretion is These results are consistent with the hypothesis, based on previous studies in the rat, that fructose-induced hyperuricemia in man results from an increased degradation of purine ribonucleotides.
H
clinical disorder. However, in many YPERURICEMIA is a common instances the precise mechanisms responsible for the elevated serum urate concentration are not well understood. Elucidation of the mechanism by which certain normal metabolic intermediates and their structural analogues alter the serum urate concentration may be useful in delineating potential pathophysiologic alterations leading to hyperuricemia. This approach proved to be valuable in defining the deficiency of hypoxanthine-guanine phosphoribosyl transferase in the Lesch-Nyhan syndrome and in some patients with gout.’ The infusion of large doses of fructose in man initiates a number of biochemical changes.“-‘” Many of these alterations, such as the development of hyperlactacidemia, the decrease in serum inorganic phosphate, and the decrease in plasma glucose can be attributed to the phosphorylation of fructose and the entrance of this phosphorylated intermediate into the glycolytic pathway. A striking increase in the serum urate concentration has also been observed following the infusion of fructose.4’g However, the mechanism responsible for this effect has been unclear. In the present study, we have confirmed the observation that fructose produces hyperuricemia and have examined the mechanism. Our data in man provide support for previous conclusions based on studies in the rat in which hyperuricemia was attributed to an accelerated catabolism of purine nucleotides.11-13 From fhe Division of Rheumntic and Genetic Diseases, Depnrfment of Medicine, Drrke University Medical Center, Durham, N. C. Received for publication January 7, 1972. Supported by USPHS Grant AM 14362. Irving H. Fox, M.D.: Postdoctornl Fellow of the Medical Research Council of Canada; Research Fellow, Division of Rheumntic and Genetic Diseases, Duke Uniuersify Medical Center, Durham, N. C. William N. Kelley, M.D.: Chief, Division of Rheumntic and Genetic Diseases, and Associate Professor of Medicine, Assistant Professor of Biochemistry, Duke University Medical Center, Durhnm, N. C. Metabolism,
Vol. 21, No. 8 (August), 1972
713
Range
M
2 1 S.D.3*
t Mean
1 S.D.3l
k
* Mean
(D.H. F7 2 420)
M
(D.H. JO1630) L.L.
M
M
J.W.
(D.H. H93284)
(D.H. J13731) H.M.
Patients A.B.
Normal
Sex
61
60
48
75
Age (Yr)
a.a+o.l
8.5k0.2
5.7kO.l
5420.4
3-7
Plasma Urate (mg/lOO ml)
ia
31
16
44
6-25
UV,,ste (mg/hr)
1. Clinical
-
33
12
35
106
145
86
142
(@moles/ C,, (ml/min) hr)
osspurines
uv
Table
5.5
5.9
4.7
13.6
C,,,t, (ml/min)
5.2
4.1
5.5
9.6
5.412.1+
C,,,,t,/C,, ( x 100)
Summary
-
0.77
-
0.86
ATP (mW .w
5.7k1.4
4.o-co.7
3.7kO.9
2.3-cO.3’
4.4&l
(,llW
-
1.4-co.4
1.420.4
-
Ribose-5-P CPM)
Concentration
PP-ribose-P
Erythrocyte
127
108
100
110
98ei4t
mg/hr)
(nmolesl
Hypoxanthine
13
29
40
31
31 i61
mg/hr)
Adenine (nmoles/
Erythrocyte Lysate Phosphoribosyltransferase Activity
STUDIES
OF FRUCTOSE-INDUCED
715
HYPERURICEMIA
MATERIALS
AND METHODS
Four male patients with gout were admitted to the Clinical Research Unit at Duke University Medical Center. The diagnosis of gout was based on the clinical history in all four subjects, the presence of tophi in patients A.B. and J.W., and the demonstration of intracelhrlar monosodium urate crystals by compensated, polarized light microscopy in synovial fluid obtained from subjects L.L. and H.M. during an acute episode of monoarticular arthritis. The patients had essentially normal renal function as measured by glomerular filtration rate. All patients had normal hypoxanthine-guanine phosphoribosyl transferase activity, and one patient was heterozygous for a deficiency of adenine phosphoribosyl transferase (Table 1). Based on the daily excretion of uric acid in the urine after equilibration on a diet essentially free of purines, patient J.W. (654 mg/24 hr) would be classed as an overexcretor, whereas H.M. (394 mg/24 hr), A.B. (441 mg/24 hr), and L.L. (424 mg/24 hr) would be considered normal excretors. The patients received a diet essentially free of purines, with 2200-2600 kcal and 70 g protein for 5 days prior to and throughout the study period. Control studies were performed at least IO days after discontinuing all drugs known to affect uric acid metabolism or excretion. All patients received colchicine, and one patient (A.B.) received quinidine throughout the study period. Studies during allopurinol therapy were performed after the administration of 600 mg/day for at least 48 hr. The patients were fasting, except for water, for 12 hr prior to each study. On the morning of the study, an infusion of isotonic saline was started. Three blood samples were obtained over a I-hr control period. Fructose (0.5 g/kg), sterilized by ultrafiltration, was infused as a 20% solution over a IO-min period. Blood samples were obtained every 15 min during the first hour after the start of the infusion, every 30 min during the second hour, and at the end of the third hour. Urine was collected by voluntary voiding at specified times. Four I-hr collection periods were utilized corresponding to the control period and the first, second, and third hours after the infusion. All blood samples were placed immediately in ice and centrifuged within 15 min at 4°C. Five milliliters of blood, collected in 0.1 ml of 0.2 M EDTA, was processed as previously described, and the extracts assayed for phosphoribosylpyrophosphate (PI’-ribose-I’)14 and ribose-5-phosphate.15 Heparinized blood for ATP assay was extracted using the perchloric acid method of Wailer et al.18 ATP was estimated by the firefly bioluminescence assay.17 Oxalated venous blood for lactate was extracted immediately at 4’C with an equal volume of 7% perchloric acid and assayed by the lactate dehydrogenase assay.18 Uric acid was measured by the uricase method19 and oxypurines were measured by the enzymatic conversion to uric acid.20 Urinary creatinine was determined by the method of Taussky.21 Total reducing sugar was measured on a SMA 6/60, while plasma creatinine and phosphorus were measured on a SMA 12160 using standard techniques. Plasma glucose was measured using the glucose oxidase method. Hypoxanthine-guanine phosphoribosy1 transferase and adenine phosphoribosyl transferase were assayed by radiochemical methods.22 Intact erythrocytes from two gouty patients, including one of the patients (L.L.) who was studied in vivo and one patient with the Lesch-Nyhan syndrome and a deficiency of hypoxanthine-guanine phosphoribosyl transferase (W.E.), were incubated for 2 hr in phosphate-buffered saline as previously described14 without glucose or fructose, with 0.13 M glucose, or with 0.13 M fructose. The erythrocytes were extracted and assayed for PI’-ribose-B.14 When statistical
possible, results significance.
were
analyzed
by
the
paired
two-tailed
Student’s
t test
of
RESULTS The in Table
baseline
data
I. The values
collected presented
in the
four
patients
were obtained
studied
over a I-hr
are
summarized
period
immediately
prior to the fructose infusion and represent the control values for Figs. I and 2. The values for plasma urate, erythrocyte PP-ribose-P, and erythrocyte
FOX AND KELLEY
716
+60r _
-100 +800
c
p\ Fig. 1. Effect of fructose infusion on plasma w-ate and urinary excretion of
uric acid and oxypurines. Results are expressed as per cent of control values L
180
TimelMlnutes)
(Table 1). Solid lines indicate no allopurinol; broken lines indicate pretreatment with allopurinol. Open squares, patient J.W.; black squares, patient L.L.; open circles, patient KM.; and black circles, patient A.B.
ribose-S-phosphate represent the mean obtained during the I-hr control period.
The infusion urate in all four after beginning two patients in therapy. Urinary
*
SD
of fructose produced a significant patients (Fig. IA). The maximum the infusion. There was no rise whom the studies were repeated
Oxypwines
and
of three
different
samples
rise ( p
Uric Acid
As illustrated in Fig. 1B, the infusion of fructose produced a substantial increase in the excretion rate of uric acid. In each patient, the maximal effect occurred in the first hour following the infusion. A modest increase in uric acid excretion was also noted when the fructose was administered to the two subjects receiving allopurinol. In each patient studied, the infusion of fructose also produced a striking increase in the excretion rate of oxypurines (Fig. 1C). The greatest changes were observed during the first hour after the infusion, with levels tapering rapidly back to the control range within 3 hr. The increase in urinary oxypurines was somewhat greater in the two subjects being treated with allopurinol prior to the fructose infusion than that observed in these same subjects before treatment with allopurinol was instituted.
STUDIES
OF FRUCTOSE-INDUCED
717
HYPERURICEMIA
The urine from two of the patients treated with fructose was subjected to anaIysis on an ultraviolet analyzer. The marked rise in urinary oxypurines following intravenous fructose infusion resulted primarily from a rise in hypoxanthine excretion. In addition, a striking increase in the excretion of inosine was also noted. These observations are consistent with the hypothesis that fructose-induced hyperuricemia results from a stimulation of nucleotide catabolism. There was no consistent change in the fractional clearance of uric acid either before or after the treatment with allopurinol (Table 2). Erythrocyte nnd ATP
Phosphovibosylpyrophosphate
(PP-ribose-P),
Ribose-5-phosphate,
As illustrated in Table 3, fructose as well as glucose is capable of stimulating PP-ribose-P production in washed erythrocytes in viva. The infusion of fructose in vivo produced a modest but significant decrease in erythrocyte PP-ribose-P levels in each patient studied (p < 0.05 at 15 and 30 min (Fig. 2). In addition, the intracellular levels of ribose-S-phosphate also began to decrease 45 min after the infusion of fructose in the two patients studied (Fig. 2). The infusion of fructose had no discernible effect on the concentration of ATP in erythrocytes during the 3-hr study period (Fig. 2). Table 2. Effect of Fructose
on Fractional
Excretion
and Clearance
(w/ml)
C “rate&r ( x 100)
0.31 0.40 0.46 0.60 0.17 0.19 0.21 0.18 0.19 0.17 0.15 0.15 0.21 0.48 0.27 0.26 0.29 0.24 0.23 0.25 0.18 0.14 0.16 0.15
9.6 9.1 11.0 14.5 5.2 4.9 5.3 4.8 5.5 4.1 3.7 3.3 4.1 7.3 4.1 4.1 6.1 7.6 7.5 8.0 5.3 4.3 4.8 4.5
U”wate/Ccr Subject
Period
Control 1 2 3 Control 1 2 3 Control 1
A.B.
L.L.
H.M.
2 3 Control 1 2 3 Control 1 2 3 Control 1 2 3
J.W.
A.B.*
J.W.’
l
Receiving
allopurinol,
600 mg/day.
of Uric Acid
718
FOX AND KELLEY
-60 Y E@ t40 2 ,” ;g 0 +E @-40 -80 -%+40 PZF GE” 0 g6 -40 0
30
60
90
120 150 180
Fig. 2. Effect of fructose infusion on concentration of PP-ribose-P, ribose-5phosphate, and ATP in erythrocytes. Results are expressed as per cent of control values (Table 1). Control values represent mean + SD (vertical bars) of three different samples obtained during a 60-min period immediately preceeding infusion of fructose. Open squares, patient J.W.; black squares, patient L.L.; open circles, patient H.M.; and black circles, patient A.B.
Time (Mtnutes)
Table 3. Effect of Glucose and Fructose on Phosphoribosylpyrophosphate Production in Human Erythrocytes In Vitro PP-ribose-P Subject
E.C. W.E. L.L.
Concentration
(pM)
Control
Glucose (0.13 U)
Fructose (0.13 M)
46 201 45
154 358 75
329 455 233
Table 4. Effect of Fructose on Blood Lactate Concentration Blood Lactate Control (mg/lOO ml)
No treatment A.B. H.L.M. J.W. Allopurinol A.B. J.B.
Other The
30 Min After Infusion (mgllO0 ml) (% Change)
6.8 8.9 4.4
28.0 22.0 18.0
+312 +147 +309
6.5 8.0
21.0 30.0
+223 +275
Plnstrm Chemistries infusion
of fructose
crease in the concentration
produced
a transient
of total reducing
the concentration of glucose during the 3-hr study period.
and phosphate
but
substances remained
striking
(fourfold)
in the plasma, essentially
in-
although unchanged
The blood lactate concentration increased markedly 30 min after infusion of fructose (Table 4). This hyperlacticacidemic effect of fructose not altered by pretreatment with allopurinol.
the was
STUDIES
OF FRUCTOSE-INDUCED
HYPERURICEMIA
719
DISCUSSION The phenomenon of fructose-induced hyperuricemia was initially described the hyperuricemic effect of in man by Perheentupa and Raivio. 4 Although fructose has not been consistently confirmed by other investigators,* there appears to be a relationship between the rate of fructose administration and the degree of hyperuricemia produced. g Our studies confirm those of others that fructose, at a dose of 0.5 g/kg infused over a IO-min period, produces a marked and consistent hyperuricemia.
Four possible mechanisms can be formulated to account for this hyperuricemic effect of fructose: (I) a shift in uric acid pool or decreased extrarenal disposal of uric acid leading to an increased filtered load, (2) decreased renal clearance of uric acid, (3) an increased rate of purine synthesis de novo, or (4) an accelerated degradation of purine ribonucleotides. Our data allow the analysis of each of these potential mechanisms. The elevation of urinary oxypurines and the inhibition of fructose-induced hyperuricemia by allopurinol eliminate a shift in the uric acid pool or an alteration of extrarenal disposal of uric acid as an explanation of the abrupt rise in plasma urate. A decreased renal clearance of uric acid would warrant consideration as the mechanism responsible for fructose-induced hyperuricemia, since most drugs that produce hyperuricemia do so by this mechanism.‘” In addition, fructose produces an increase in the plasma lactate concentration,‘-’ and lactic acid itself decreases the renal clearance of uric acid.“” However, the infusion of fructose produced no consistent decrease in the fractional clearance of uric acid. The possibility that fructose may cause hyperuricemia by increasing the rate of purine biosynthesis de novo was considered. As demonstrated in the present study, fructose is both a precursor and stimulator of PI’-ribose-I’ synthesis in human cells in vitro. Since PP-ribose-P is a limiting substrate for the initial step of purine biosynthesis de novo,05’06 an increased concentration of PI’-ribose-P would be expected to increase the rate of purine synthesis de novo. However, several findings indicate that the rapid increase in plasma urate concentration, which occurs 30-45 min after the infusion of fructose, does not appear to be due to this mechanism. (1) The infusion of fructose did not increase PP-ribose-P levels in erythrocytes in vivo; in fact, the intracellular concentration of PP-ribose-P, as well as its immediate precursor, ribose-5-phosphate, appeared to decrease. (2) Others have demonstrated that hyperuricemia occurs following the infusion of fructose to patients with hereditary fructose intolerance in whom there is a genetically determined block in the further metabolism of fructose-l-phosphate.4 This finding also suggests that increased uric acid production can occur without stimulating PI’-ribose-P synthesis. (3) It seems unlikely that an increase in purine biosynthesis de IIOVO would lead to so rapid an increase in the serum urate concentration. The most potent stimulator of purine biosynthesis de novo known in man, 2-ethylamino-1, 3,4-thiadiazole, increases the serum urate concentration in 24-48 hr, not in 30-45 min.37.“s Our observations are most consistant with the hypothesis that fructose
FOX AND KELLEY
720
leads to a rapid degradation of purine nucleotides with the consequent formation of inosine, hypoxanthine, xanthine, and finally uric acid. Recent studies in the rat in vivo, in perfused rat liver, and in human liver have demonstrated that fructose decreases the concentration of adenine nucleotides, especially ATP, and inorganic phosphate within the ce11.11-132”g Since ATP normally inhibits S-nucleotidase and inorganic phosphate inhibits AMP deaminase, these changes would be expected to stimulate the catabolism of AMP to inosine.13 This hypothesis would readilv account for the rapid rise in serum urate concentration and in the urinary excretion of oxypurines and uric acid following the infusion of fructose. The decreased ervthrocyte PPribose-P levels, which occurred prior to the decrease in ribose-s-phosphate concentration, may reflect PP-ribose-P consumption perhaps due to an increased reutilization of hypoxanthine. Only our finding that erythrocyte ATP levels remain unchanged following the infusion of fructose is difficult to reconcile with this hypothesis. However, the concentration of ATP in erythrocytes is normally 400-800 times higher than the concentration of PP-ribose-P and ribose-5-phosphate; it is likely that changes in ATP concentration of the order of magnitude of those observed for PP-ribose-I’ or ribose-5-phosphate would not be detected. Emmerson has recently demonstrated that the chronic oral administration of fructose to three patients led to a small increase in the incorporation of glycine-l-l4 into uric acid in vivo.30 Although this observation is consistent with the hypothesis that fructose increases purine biosynthesis de novo, it is equally consistent with the hypothesis that over a period of days the depletion of purine nucleotides would result in a release of feedback control on purine biosynthesis de novo. ACKNOWLEDGMENT The authors wish to thank Mrs. Dr. J. B. Sidbury, Jr., for measuring McManus for measuring erythrocyte
Margaret Evans for her careful technical assistance, glucose by the glucose oxidase method, and Dr. T. J. ATP concentrations.
REFERENCES 1. Kelley, W. N., Greene, M. L., Rosenbloom, F. M., Henderson, J. F., and Seegmiller, J. E. : Hypoxanthine-guanine phosphoribosyltransferase deficiency in gout. Ann. Intern. Med. 70:155, 1969. 2. Miller, M., Craig, J. W., Drucker, W. R., and Woodward, H.: The metabolism of fructose in man. Yale J. Biol. Med. 29:335, 1956. 3. Froesch, E. R. : Essential fructosuria and hereditary fructose intolerance. In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S. (Eds.): The Metabolic Basis of Inherited Disease. 1966, p. 124. 4. Perheentupa, J., and Raivio, K.: Fructose-induced hyperuricemia. Lancet 2:528, 1967. 5. Bergstrom, J., and Hultman, E.: Syn-
thesis of muscle glycogen in man glucose and fructose infusion. Acta Stand. 182:93, 1967.
after Med.
6. -, -, and Roth-Norlund, A. E.: Lactic acid accumulation in connection with fructose infusion. Acta Med. Stand. 184 :359, 1968. 7. Levin, B., Snodgrass, J. A. I., Oberholzer, V. G., Burgess, E. A., and Dobbs, R.: Fructosaemia, observations of seven cases. Amer. J. Med. 45~826, 1968. 8. Sahebjami, H., and Scalettar, R.: Effects of fructose infusion on lactate and uric acid metabolism. Lancet 1:366, 1971. 9. Heuckenkamp, P-U, and Zollner, N.: Fructose-induced hyperuricaemia. Lancet 1 :808,1971.
STUDIES
OF FRUCTOSE-INDUCED
HYPERURICEMIA
10. Morris, R. C.: An experimental renal acidification defect in patients with hereditary fructose intolerance I. Its resemblance to renal tubular acidosis. J. Clin. Invest. 47:1359, 1968. 11. Maenpaa, I’. H., Raivio, K. O., and Kekomaki, M. I’. : Liver adenine nucleotides: Fructose-induced depletion and its effect on protein synthesis. Science 161:1253, 1968. 12. Raivio, K. O., Kekomaki, M. I’., and Maenpaa, I’. H.: Depletion of liver adenine nucleotides induced by D-fructose. Biochem. Pharmacol. 18:2615, 1968. 13. Woods, H. F., Eggleston, L. V., and Krebs, H. A.: The cause of hepatic accumulation of fructose-l-phosphate on fructose loading. Biochem. J. 119501, 1970. 14. Fox, I. H., Wyngaarden, J. B., and Kelley, W. N.: The depletion of erythrocyte phosphoribosylpyrophosphate by allopurinol in man. New Eng. J. Med. 283:1177,1970. 15. -, and Kelley, W. N.: Human phosphoribosylpyrophosphate synthetase: Kinetic mechanism and end-product inhibition. J. BioI. Chem. 247:2126, 1972. 16. Waller, H., Lohr, G., and Tabatabai, M.: Hamolyse und sehlen zon glucose-6phosphatdehydrogenase n roten blutzellen. Klin. Wschr. 35 :1022, 1957. 17. McManus, T. J., Allen, D. W., and Hyun, D. K.: Unpublished observations. 18. Ohlson, G. F.: Optimal conditions for the enzymatic determination of L-lactic acid. Clin. Chem. 8:1, 1962. 19. Liddle, L., Seegmiller, J. E., and Laster, L.: The enzymatic spectrophotometric method for determination of uric acid. J. Lab. Clin. Med. 54:903, 1959. 20. Klinenberg, J. R., Goldfinger, S., Bradley, K. H., and Seegmiller, J. E.: An enzymatic spectrophotometric method for the determination of xanthine and hypoxanthine. Clin. Chem. 13834, 1967. 21. Taussky, H. H.: A microcolorimetric determination of creatinine in urine by the Jaffe reaction. J. Biol. Chem. 208:853, 1954. 22. Kelley, W. N., Rosenbloom, F. M., Henderson, J. F., and Seegmiller, J. E.: A
721
specific enzyme defect in gout associated with overproduction of uric acid. Proc. Nat. Acad. Sci. USA 57:1735, 1967. 23. Wyngaarden, J. B., and KelIey, W. N.: Gout. The Metabolic In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S. (Eds.): The Metabolic Basis of Inherited Disease (ed. 3). New York, McGraw-Hill, 1972. 24. Howell, R. R., Ashton, D. M., and Wyngaarden, J. B. : Glucose-6-phosphate deficiency glycogen storage disease. Studies on the interrelationships of carbohydrate, lipid and purine abnormalities. Pediatrics 29 :553, 1962. 25. Kelley, W. N., Fox, I. H., and Wyngaarden, J. B.: Essential role of phosphoribosylpyrophosphate in regulation of purine biosynthesis in cultured human fibroblasts. Clin. Res. 18:457, 1970. 26. -, Greene, M. L., Fox, I. H., Rosenbloom, F. M., Levy, R. I., and Seegmiller, J. E.: Effects of erotic acid on purine and lipoprotein metabolism in man. Metabolism 19 :1025, 1970. 27. Krakoff, I. H., and Balis, M. E.: Studies of 2-substituted thiadiazoles in man. J. Clin. Invest. 38 :907, 1959. 25. Seegmiller, J. E., Grayzell, A. I., Liddle, L., and Wyngaarden, J. B.: The effect of ?=ethylamino-1,3,&thiadiazole on the incorporation of glycine into urinary purines and uric acid in man. Metabolism 12:507, 1963. 29. Bode, L., Shumacher, H., Goebell, H., Zelder, O., and Pelzel, H.: Fructose-induced depletion of liver adenine nucleotides in man. Hormone Metab. Res. 3:71, 1971. 30. Emmerson, B.: Personal communication. 31. Smith, M. L., and Scott, J, T.: Uric acid clearance in patients with gout and normal subjects. Ann Rheum. Dis. 30:285, 1970. 32. Fox, I. H., and Kelley, W. N.: Phosphoribosylpyrophosphate in man : Biochemical and clinical significance. Ann. Intern. Med. 74~424, 1971.