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PubMedDate not supplied

Postsecretory reabsorption of urate in man.

These results are consistent with a model for renal tubular transport of urate in which there is reabsorption of both filtered and secreted urate. Urate secretion greatly exceeds total urate excretion, and most secreted urate is reabsorbed. At least a portion of urate reabsorption occurs at a site distal to or coextensive with the urate secretory site. There appear to be at least two distinct reabsorptive mechanisms for urate. The results of the flow rate and vasopressin studies are consistent with the hypothesis that urate reabsorption occurs in both the distal and the proximal tubule in man. The distal reabsorptive site appears to be quite small. It may be passive since it does not appear to be inhibited by uricosuric drugs. This reabsorptive site may account for less than 15% of total urate reabsorption. Both volume expansion and probenecid may inhibit urate absorption only in the proximal tubule. Thus reabsorption in the proximal tubule coud account for more than 90% of total urate reabsorption. Reabsorption at the postulated collecting duct reabsorptive site appears to be too small in magnitude to account for all reabsorptions of secreted urate. This could be explained if the reabsorptive site in the proximal tubule is coextensive with or distal to the secretory site. Alternatively, there might be two reabsorptive sites in the proximal tubule: a presecretory site accounting for the reabsorption of most filtered urate, and a site either coextensive or distal to the secretory site accounting for a major component of reabsorption of secreted urate. Finally urate reabsorption would also take place in the collecting duct, perhaps at a passive, flow-dependent site.

Aspirin↗
PubMed1996

The effect of mannitol and secretin on the biliary transport of urate in humans.

No evidence is available on the transport of biliary urate and the possible role of choleretic agents in the regulation of biliary urate elimination in humans. To test this hypothesis we studied the following: (1) 45 cholecystomized patients to determine urate levels in hepatic bile and gallbladder bile, and (2) 13 cholecystomized patients fitted with T-tubes to determine the effects of secretin injection (either 70 U of porcine secretin or 0.02 mg.kg-1 of synthetic human secretin, as a single dose) and/or mannitol infusion (5 cm3.min-1 for 90 minutes) on biliary urate excretion. In the latter group, samples of bile and serum were analyzed for urate under basal state and after the administration of both agents. In our first approach, results showed that urate concentrations present in hepatic as well as in gallbladder bile were much lower than the corresponding values in serum (P < .001). The mean gallbladder bile urate concentration was not significantly increased over the concentration in hepatic bile. When compared with basal state values, procine and synthetic secretin induced a significant increase in mean urate clearance (P < .001) because of a significant increase in mean bile flow (P < .001), whereas the mean biliary urate concentration significantly decreased (P < .001) with a concomitant decrease in the mean serum urate concentration (P < .02). Mannitol also induced a significant increase in the mean urate clearance (P < .002) because of a significant increase in the mean biliary urate concentration (P < .01) with a concomitant decrease in the mean serum urate concentration (P < .01) and without changes in the mean bile flow (P > .05). Therefore, it appears that a substantial amount of urate is eliminated by biliary route. The load of biliary urate excreted may be modified by mannitol and secretin and possibly other factors, a finding that could have an application in some pathological conditions associated with decreased renal urate excretion.

Adult↗
PubMed1981

Nature of urate transport in isolated rabbit proximal tubules.

Unidirectional fluxes of [14C]-urate from bath to lumen and from lumen to bath were measured in isolated perfused rabbit proximal tubules. The absorption of urate from the perfusate was small in magnitude and relatively insensitive to alteration by luminal probenecid or D-glucose. By contrast, peritubular probenecid inhibited urate secretion from bath to lumen in all three segments. To estimate the facilitated component of urate secretion for each bath concentration of the anion, the passive component of urate influx (probenecid-resistant influx) was subtracted from the total influx of urate. Urate secretion was strongly dependent on concentration of urate in the bath, the relationship being sigmoidal in many S1 and S2 segments. The S0.5 values of 187,251, and 234 X 10(-6) M obtained from Hill plots of mean data from S1, S2, S3 segments, respectively, indicated that the secretory mechanism had a relatively uniform affinity for urate along the proximal tubule. Vmax values for urate secretion were 568,592, and 55 X 10(-15) mol . min-1 . mm-1 for the same segments. Kinetic analysis indicated that axial heterogeneity of urate secretion is probably due to differences in the relative densities of urate transporters of common affinity along the proximal tubule. We suggest that in intact rabbits with low plasma levels urinary urate is the product of glomerular filtration, passive reabsorption, and minimal proximal secretion. With elevation of plasma urate above endogenous levels, S1 and S2 segments secrete urate at an accelerated rate, possibly reflecting action of a transporter with allosteric features.

Animals↗
PubMed2002

Environmental lead exposure and urate excretion in the general population.

PURPOSE: Chronic occupational exposure to lead is related to low urate excretion and a high incidence of gout in lead workers. However, whether chronic low-level environmental lead exposure influences urate excretion in the general population remains unknown. SUBJECTS AND METHODS: We studied 111 healthy subjects with normal renal function (serum creatinine level < or =1.4 mg/dL) and no previous lead exposure or systemic diseases. All subjects had their blood lead levels measured, received ethylenediaminetetraacetic acid mobilization tests to assess their body lead burdens, and were investigated for renal function and urate excretion to assess the relation between lead and urate excretion. We studied urate excretion before and after lead chelation therapy in 24 subjects with high-normal body lead burden (>78 mg and <600 mg). RESULTS: Healthy subjects with gout (n = 27) manifested a higher body lead burden (84 +/- 42 mg vs. 45 +/- 30 mg, P <0.0001) and lower urate clearance (3.7 +/- 1.2 mL/min/1.73 m(2) vs. 6.0 +/- 2.8 mL/min/1.73 m(2), P <0.0001) than did those without gout (n = 84). Blood lead levels and body lead burden of all subjects were within the safe range. In analyses that adjusted for age, sex, body mass index, protein intake, and creatinine clearance, blood lead level was significantly related to serum urate level (beta coefficient [+/- SE] = 0.23 +/- 0.11, P = 0.03), and body lead burdens were related to all indices of urate excretion (serum urate: beta coefficient = 0.023 +/- 0.005, P <0.0001; daily urate excretion: beta coefficient = -1.55 +/- 0.40, P = 0.0002; urate clearance: beta coefficient = -0.030 +/- 0.006, P <0.0001; fractional urate excretion: beta coefficient= -0.034 +/- 0.006, P <0.0001). Following lead chelation therapy, urate clearance increased after body lead burden was reduced (3.4 +/- 1.2 mL/min/1.73 m(2) vs. 4.9 +/- 1.4 mL/min/1.73 m(2), P <0.005). CONCLUSION: Chronic low-level environmental lead exposure may inhibit urate excretion in the general population, and lead chelation therapy reduces this inhibition. These findings support efforts to reduce sources of environmental lead exposure and suggest alternative approaches to hyperuricemia and gout in the general population.

Adult↗
PubMed1983

Mechanism of urate and p-aminohippurate transport in rat renal microvillus membrane vesicles.

The transport of urate and p-aminohippurate (PAH) was evaluated in brush border membrane vesicles from the rat renal cortex. The binding of urate to the membranes was 6% of total uptake and no conversion of urate to allantoin was detected. The binding of PAH to the membranes was 24% of total uptake. In the presence of an outwardly directed hydroxyl ion gradient (pHi = 7.5, pHo = 6.0), the uptake of urate and PAH was stimulated relative to the absence of a hydroxyl ion gradient (pHi = pHo = 7.5) and the influx of urate resulted in a transient overshoot of the equilibrium value. The hydroxyl ion gradient-stimulated uptake of urate and PAH was not solely due to a change in membrane potential. Probenecid, DIDS, furosemide, and pyrazinoate inhibited the hydroxyl ion gradient-stimulated uptake of urate and PAH in a dose-dependent manner. The uptake of [14C]urate and [3H]PAH could be cis-inhibited and trans-stimulated by either unlabeled urate or PAH. In the presence of an outwardly directed bicarbonate gradient and 10% CO2 (outside HCO-3 = 5.4 mM, inside HCO-3 = 54 mM, pHo = 6.5, pHi = 7.5), the initial rate of urate uptake was faster and the initial rate of urate efflux was slower compared with vesicles that had the same pH gradient without bicarbonate or CO2. The effects of bicarbonate gradients on organic anion transport were not dependent on diffusion potentials. Finally, 100 mM extravesicular Na+, K+, Li+, or Cs+ did not affect urate or PAH uptake. These results indicate that brush border membrane vesicles from the rat kidney contain an anion exchange transport system with affinity for urate, PAH, hydroxyl ions, and bicarbonate. In addition there is no evidence for a sodium-urate or sodium-PAH cotransport mechanism in these membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1976

Renal transport of urate during diuretic-induced hypouricemia.

The effect of two weeks administration of a uricosuric diuretic (SKF-62698) on renal urate handling has been examined in 11 normal men. Plasma urate concentrations had declined by more than 60 per cent after two weeks. Urate excretion per unit of glomerular filtration rate and urate clearance (Curate) per unit of glomerular filtration rate were increased after the administration of SKF-62698. The importance of intact tubular secretion of urate in producing these changes was assessed by administering pyrazinamide, an agent that curtails urate secretion, to each participant. The decrements in urate excretion and clearance produced by pyrazinamide both increased significantly, whereas the residual urate excretion rates and clearances not suppressible by pyrazinamide were only minimally altered by SKF-62698 treatment. These results suggest that the excretion of secreted urate was enhanced by prolonged administration of SKF-62698, probably secondary to the inhibition of postsecretory urate reabsorption. In addition, because the nonsuppressible urate excretion did not decline despite a 63 per cent reduction in the plasma urate, it is likely that the reabsorption of filtered urate also was impaired by SKF-62698.

Adult↗
PubMed1981

Urate transport by the isolated perfused S2 segment of the rabbit.

Urate transport was studied in isolated perfused S2 segments of the superficial proximal tubule of the rabbit. When urate was present in identical concentrations of 290 microM in the perfusing and bathing solutions, there was a net secretory flux of urate of 775.0 +/- 152.8 fmol.min-1.mm-1. When urate was present in varying concentrations in the bathing solution only, the bath-to-lumen flux of urate increased as the concentration of urate in the bathing solution was increased from 60 to 595 microM, but tended toward a plateau at higher concentrations. After correction of the net secretory flux for the contribution of passive permeation, an apparent Km of 238 microM and Vmax of 950 fmol.min-1.mm-1 for the secretory flux of urate was calculated. When urate was present only in the luminal perfusion solution, the lumen-to-bath flux of urate was significantly less than the bath-to-lumen flux at a similar bath concentration of urate. The lumen-to-bath flux of urate was not significantly affected by cooling the bath to 25 degrees C. These studies provide evidence for both passive and facilitated mechanisms for urate secretion in the rabbit S2 segment. The absorptive flux for urate, however, appears to be primarily a passive mechanism.

Animals↗
PubMed1985

Urate and p-aminohippurate transport in rat renal basolateral vesicles.

We examined the transport of urate in basolateral membrane vesicles from the rat kidney and determined the relationship between the transport of urate and p-aminohippurate (PAH). Urate was not converted to allantoin, and the uptake of urate represented transport into an osmotically active intravesicular space. The 10-s uptake of 53 microM [14C]urate in basolateral vesicles was inhibited 39 +/- 6, 49 +/- 10, and 35 +/- 3% by (in mM) external 2.4 probenecid, 2.4 DIDS, and 1.4 unlabeled urate, respectively. The 10-s uptake of 353 microM [14C]urate was trans-stimulated 82 +/- 8% by preloading basolateral vesicles with 1.5 mM unlabeled urate. The uptake of urate was stimulated by an outwardly directed gradient for Cl- (Cl-in = 25 mM, Cl-out = 5 mM). This effect was not consequent to a more electropositive intravesicular space, as monitored by the voltage-sensitive sodium-L-malate cotransport system. The Cl- gradient-stimulated component of urate uptake in basolateral vesicles was not cis-inhibited by 4.8 mM PAH, whereas Cl gradient-stimulated urate uptake in brush border vesicles was cis-inhibited 43 +/- 5% by PAH. In the absence of Cl-, 4.8 mM PAH did not cis-inhibit, and 5.4 mM PAH or 6.4 mM lactate did not trans-stimulate the uptake of urate in basolateral vesicles, contrasting with results obtained with brush border vesicles. The uptake of urate in basolateral vesicles was not stimulated by external Na+ relative to K+, Li+, or Cs+. In contrast, PAH uptake in basolateral vesicles was stimulated 87 +/- 9% by external Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Lipoproteins containing apoprotein B are a major regulator of neutrophil responses to monosodium urate crystals.

The inflammatory response to intraarticular urate crystals is known to be variable in gouty arthritis. One source of variability may be the modulation of cellular responses by crystal-bound proteins. We have identified three apolipoproteins among the polypeptides bound to urate crystals exposed to plasma. Identification was first based on their coelectrophoresis with polypeptides from isolated lipoproteins and diminution in the protein coat of crystals exposed to lipoprotein-depleted plasma. The apoproteins were immunochemically identified by the Western blotting technique as apoprotein A-I, apoprotein B (apo B), and apoprotein E. Because neutrophils play a central role in acute gout, we investigated the potential effects of lipoproteins on neutrophil-urate crystal interactions. Plasma profoundly inhibited urate crystal-induced neutrophil luminol-dependent chemiluminescence (CL). Lipoprotein depletion by KBr density gradient centrifugation completely abrogated the inhibitory effect of plasma on urate-induced CL. The inhibitory activity of lipoprotein-depleted plasma was restored by adding back the d less than or equal to 1.25 g/cm3 lipoprotein fraction. Plasma also inhibited urate crystal-induced neutrophil superoxide generation and cytolysis (lactic dehydrogenase loss). This inhibition was significantly diminished by lipoprotein depletion, indicating that the lipoprotein effect was not limited to CL. Lipoprotein-depleted plasma reconstituted with very low, intermediate, and low density lipoproteins (LDL) inhibited crystal-induced CL. High density lipoprotein reconstitution was without effect. Immunodepletion from plasma of all apo B lipoproteins by agarose-bound apo B-specific antibody also removed all inhibitory activity for urate-induced CL. Thus, apo B lipoproteins were shown to be the inhibitory species in plasma. Binding of apo B lipoproteins to urate crystals and inhibition of CL was also seen in the absence of other plasma proteins. In addition, the binding of whole lipoprotein particles to the crystals was verified by detection of crystal-associated cholesterol in addition to the apoprotein. The effects of LDL on urate crystal-induced CL were stimulus specific. Coincubation of urate crystals and neutrophils in the presence of 10 micrograms/ml LDL resulted in 83% inhibition. In contrast, CL responses to a chemotactic hexapeptide, opsonized zymosan, and Staphylococcus aureus were not inhibited by LDL. The effects of depletion of apo B lipoproteins on plasma suppression of urate crystal-induced CL appeared to be unique. Plasma or sera depleted of other urate crystal-binding proteins including fibrinogen, fibronectin, C1q, and IgG retained virtually all their CL inhibitory activity. Lipoproteins containing apo B are thus a major regulator of neutrophil responses to urate crystals. These lipoproteins are present in variable concentration in synovial fluid and may exert an important influence on the course of gout.

Antibodies, Monoclonal↗
PubMed1989

Differences in urate metabolism between normouricemia and hyperuricemia in coronary heart disease in man.

We examined hyperuricemia in patients with coronary heart disease. In 85 patients with coronary sclerosis confirmed by coronary angiography, the serum urate level (6.08 +/- 1.60 mg/dL) was not different from that in subjects with normal coronary arteries (6.47 +/- 1.69 mg/dL). The incidence of hyperuricemia in patients with coronary sclerosis was 26%, and was significantly correlated with diuretics, obesity and hypertriglyceridemia, but not with hypertension or hypercholesterolemia. To elucidate the mechanism of urate metabolism in coronary sclerosis, we separated coronary sclerosis patients without complicating factors into hyperuricemics and normouricemics, and studied urate metabolism in comparison with subjects with normal coronary arteries. We found that normouricemics with coronary sclerosis had decreases in the filtered urate load and urate clearance with a normal urate-creatinine clearance ratio. Hyperuricemics with coronary sclerosis had decreases in urate clearance and urate-creatinine clearance ratios, but the filtered urate load was similar to that in normouricemics. It is suggested that in coronary sclerosis patients, normouricemics had a low glomerular filtration of urate with normal tubular urate transport, whereas hyperuricemics had enhanced tubular reabsorption of urate without any difference of urate filtration from normouricemics.

Coronary Disease↗
PubMed1980

Renal handling of urate in healthy man in hyperuricaemia and renal insufficiency: circadian fluctuation, effect of water diuresis and of uricosuric agents.

To differentiate between extrarenal and renal causes of hyperuricaemia and gout, clearances of urate and creatinine were monitored for 3 1/2 days in fifty-two individuals (seven with a history of gout) with no gross impairment of renal function (creatinine clearance 52-137 ml/min). Dietary purine intake was kept constant. Monophasic circadian fluctuations of fractional urate excretion (= urate clearance over creatinine clearance) were observed with peak values in the afternoon, about 50% higher than during the night. Circadian fluctuations of urinary flow rate were almost identical. However, enhancement of urinary flow rate due to water diuresis had no effect on urate clearance. Despite wide variation of plasma urate concentrations among different individuals (+/- 30% SD), daily urate excretion varied little (+/- 4% SD) and did not correlate with plasma urate (r = 0.03). Thus extrarenal factors appear not to account for the occurrence of hyperuricaemia in these patients. In contrast, a clearcut negative correlation was apparent between plasma urate concentration and fractional urate clearance (r = 0.72), which could fully account for the variations of plasma urate concentration. To elucidate further the mechanism responsible for antiuricosuria in hyperuricaemic patients, the effects of the uricosuric agents benzbromarone and probenecid were tested. A clearcut correlation was apparent between control fractional urate excretion and uricosuric effect of both benzbromarone and probenecid (r = 0.83 and 0.88, respectively), suggesting that anti-uricosuria was due to defective secretion. In an additional series, the uricosuric effect of probenecid was tested in ten patients with renal insufficiency. In these patients the uricosuric effect was clearly blunted, indicating that urate reabsorption is reduced in renal insufficiency.

Adult↗
PubMed2002

Facilitated diffusion of urate in avian brush-border membrane vesicles.

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward alpha-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.

Animals↗
PubMed1985

Urate transport in the proximal tubule: in vivo and vesicle studies.

The transport of urate in the mammalian nephron is largely confined to the proximal tubule. Depending on the species, net reabsorption or net secretion is observed. The rat, like the human and the mongrel dog, demonstrates net reabsorption of urate and has been the most extensively studied species. The unidirectional reabsorption and secretion of urate in the rat proximal tubule occur via a passive and presumably paracellular route and by a mediated transcellular route. The reabsorption of urate, and possibly its secretion, can occur against an electrochemical gradient. A variety of drugs and other compounds affect the reabsorption and secretion of urate. The effects of these agents depend on their site of application (luminal or blood), concentration, and occasionally their participation in transport processes that do not have affinity for urate. Recent studies with renal brush border and basolateral membrane vesicles from the rat and brush border vesicles from the dog have determined the mechanisms for urate transport across the luminal and antiluminal membranes of the proximal tubule cell. Brush border membrane vesicles contain an anion exchanger with affinity for urate, hydroxyl ion, bicarbonate, chloride, lactate, p-aminohippurate (PAH), and a variety of other organic anions. Basolateral membrane vesicles contain an anion exchanger with affinity for urate and chloride but not for PAH. Both membrane vesicle preparations also permit urate translocation by simple diffusion. A model for the transcellular reabsorption and secretion of urate in the rat proximal tubule is proposed. This model is based on the vesicle studies, and it can potentially explain the majority of urate transport data obtained with in vivo techniques.

Absorption↗
PubMed1982

Renal handling of urate in patients with calcium stone disease.

The renal handling of urate was investigated in 28 recurrent calcium stone formers and 12 healthy controls. 12 patients were hyperuricosuric and 16 patients had incomplete proximal or distal acidification defects. Measurements of the glomerular filtration and the tubular reabsorption of filtered urate were made through the pyrazinamide (PZA)-suppression test of urate secretion. Hyperuricosuria could not be explained by defects in the renal handling of urate. Patients with proximal acidification defects had a higher tubular reabsorption of filtered urate than the other subjects. Tubular reabsorption of filtered urate was inversely correlated, and filtered urate escaping reabsorption positively correlated to the 24-hour excretion of urate. In contrast, the PZA-suppressible fraction of urate excretion, which is the net effect of tubular secretion and post-secretory reabsorption and thought to be the main regulator of urate excretion, was not correlated to the 24-hour excretion of urate. It is concluded that the renal handling of urate is basically normal in calcium stone disease, whereas minor deviations may co-exist with renal tubular acidification dysfunction.

Calcium↗
PubMed1992

Renal handling of urate in two patients with hyperuricemia and primary hyperparathyroidism.

Two patients with primary hyperparathyroidism had hyperuricemia due to the decrease in urate clearance. In analysis by 4-component model system, the tubular secretion of urate commonly decreased without changes in either filtered urate or presecretory reabsorption of urate. Both patients had a reduction of urea clearance, and both parathyroidectomy in the former case and intravenous infusion of saline in the latter case could reduce the serum urate level associated with the increase in the ratio of urate clearance to creatinine clearance. It is of interest that the former case with a higher serum urate level had a relatively higher postsecretory reabsorption, even with the decrease in tubular secretion of urate. However, the latter patient with a lower serum urate level had a decrease in postsecretory reabsorption of urate in proportion to the decrease in tubular secretion. These results suggest that in hyperuricemia patients with primary hyperparathyroidism, the reduction of tubular urate secretion via hypoperfusion of the capillary network is typically present, however, the severity of the hyperuricemia might be dependent on the dysfunction of the postsecretory reabsorption of urate.

Aged↗
PubMed1987

Pharmacological evaluation of urate renal handling in humans: pyrazinamide test vs combined pyrazinamide and probenecid administration.

Uricosuric and antiuricosuric drugs have been utilised widely for the study of tubular urate transport in humans. A normal suppression of urate excretion after pyrazinamide is usually taken as evidence of normal presecretory reabsorption. However, in patients with reduced presecretory reabsorption, during pyrazinamide inhibition of urate secretion unreabsorbed urate might still undergo reabsorption along postsecretory sites, allowing for a normal pyrazinamide suppression of urate excretion. To test this possibility, we have performed the pyrazinamide test both alone and after pretreatment with probenecid, which should block postsecretory urate reabsorption. The test was performed in 8 controls, in 9 patients with 'low-excretory' hyperuricaemia, and in 7 patients with tubular urate wasting. Pyrazinamide-non-suppressible urate excretion after pretreatment with probenecid did not differ from the excretion obtained after pyrazinamide alone in hyperuricaemic patients (mean difference 1.33 +/- 2.3% of filtered urate; P = NS); it was slightly higher in controls (3.4 +/- 3.4; P less than 0.05), but was much higher in patients with tubular urate wasting (19.6 +/- 12.7; P less than 0.005). The pyrazinamide test, performed alone, was normal in three patients with tubular urate wasting, but it was abnormal in all patients after pretreatment with probenecid. These results are consistent with the possibility that, during maximal pyrazinamide effect, some uric acid escaping reabsorption at presecretory sites may undergo reabsorption along postsecretory sites, leading to a quantitative overestimation of presecretory reabsorption. This phenomenon appears to have clinical relevance, especially in patients with abnormal urate reabsorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Rat urate oxidase produced by recombinant baculovirus expression: formation of peroxisome crystalloid core-like structures.

Urate oxidase (EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but absent in humans and hominoid primates. In rats and most other mammals that catabolize uric acid to allantoin, this enzyme is localized within the crystalloid cores of peroxisomes present in liver parenchymal cells. To determine whether urate oxidase forms these crystalloid cores or whether core-forming protein(s) exist in association with urate oxidase, a baculovirus expression vector system was used to overproduce the full-length rat urate oxidase in Spodoptera frugiperda cells. Urate oxidase was expressed to a level of approximately 30% of the total protein in this system. Immunoblot analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties similar to those of urate oxidase expressed in rat liver. Immunofluorescence and electron microscopic examination revealed that the overexpressed recombinant urate oxidase is present in both the cytoplasm and the nucleus of infected insect cells as numerous 1- to 3-microns discrete particles. These insoluble protein aggregates, which were positively stained for urate oxidase by protein A-gold immunocytochemical approach, did not appear to be delimited by a single membrane. They revealed a crystalloid structure reminiscent of rat peroxisomal core consisting of bundles of tubules with an inner diameter of approximately 50 A. The recombinant urate oxidase particles, isolated by a single-step procedure, were composed entirely of 35-kDa urate oxidase subunit. These studies indicate that rat urate oxidase is capable of forming insoluble crystalloid core-like structures.

Animals↗
PubMed1988

Tissue specificity and species differences in the distribution of urate oxidase in peroxisomes.

The localization of urate oxidase in different tissues of rat and in the livers of selected mammalian species was investigated by immunoblot analysis and protein A-gold immunoelectron microscopy. Urate oxidase was purified from rat liver and used as an antigen to generate polyclonal antibodies in the rabbit. The antibodies were found to be monospecific by immunodiffusion and immunoblot analyses. By immunoblot analysis, urate oxidase was detected in the livers of rat, two strains of mice, hamster, dog, cat, and cow, but not in the Cynomolgus monkey and human liver. Urate oxidase was not detected by immunoblot method in rat kidney, jejunal mucosa, adrenal gland, testis, and pancreas. The subcellular localization of urate oxidase was ascertained by the protein A-gold immunocytochemical staining of the Lowicryl K4M embedded tissues. Urate oxidase was localized exclusively in the crystalloid core of the peroxisome in hepatic parenchymal cells of rat, mouse, hamster, dog, cat, and cow. The limiting membrane and the matrix of hepatic peroxisomes in these species were negative for the staining. The marginal plates of feline, canine, and bovine hepatic peroxisomes were also negative for urate oxidase. This enzyme was also not detected within the peroxisomes of human and monkey livers by the immunocytochemical technique. Peroxisomes (microperoxisomes) in extrahepatic rat tissues did not stain positively for urate oxidase by the protein A-gold immunocytochemical method, although they were positive for catalase. Fatty acyl-CoA oxidase was present in peroxisomes of jejunal mucosa, Leydig cells of test-is and pancreas but not in adrenal gland. Administration of a hepatic peroxisome proliferator, ciprofibrate or Wy-14643, failed to induce urate oxidase in rat liver. These results indicate that urate oxidase is a liver specific protein in rat and its localization within the liver peroxisomes of six mammals, excluding man and a nonhuman primate, and that its localization is limited exclusively to the crystalloid core. Unlike fatty acyl-CoA oxidase, urate oxidase does not appear to be inducible significantly by peroxisome proliferator treatment in the rat liver.

Animals↗