Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.     

Preconditioning with hydrogen peroxide (H2O2) or ischemia in H2O2-induced cardiac dysfunction

To assess whether allantoin levels in serum and urine are influenced by exhaustive and moderate exercise and whether allantoin is a useful indicator of exercise-induced oxidative stress in humans, we made subjects perform exhaustive and moderate (100% and 40% VO₂max) cycling exercise and examined the levels of allantoin, thiobarbituric acid reactive substances (TBARS) and urate in serum and urine. Immediately after exercise at 100% VO₂max, the serum allantoin/urate ratio was significantly elevated compared with the resting levels while the serum urate levels was significantly elevated 30 min after exercise. The serum TBARS levels did not increase significantly compared with the resting levels. Urinary allantoin excretion significantly increased during 60 min of recovery after exercise, however, urinary urate excretion decreased significantly during the same period. The urinary allantoin/urate ratio also rapidly increased during 60 min of recovery after exercise. Urinary TBARS excretion decreased during the first 60 min of the recovery period and thereafter significantly increased during the latter half of the recovery period. On the contrary, after 40% VO₂max of exercise, no significant changes in the levels of urate, allantoin and TBARS in serum or urine were observed. These findings suggest that allantoin levels in serum and urine may reflect the extent of oxidative stress in vivo and that the allantoin which appeared following exercise may have originated not from urate formed as a result of exercise but from urate that previously existed in the body. Furthermore, these findings support the view that allantoin in serum and urine is a more sensitive and reliable indicator of in vivo oxidative stress than lipid peroxidation products measured as TBARS.     

Urate transport in human red blood cells. Activation by ATP.

Urate transport in human erythrocytes were measured and compared to previous observations by other authors regarding inorganic anions, especially chloride. Conclusions wwere as follows: 1. Urate influx as a function of increasing concentrations showed saturation kinetics. 2. The effects of pH and of several passive anion transport inhibitors such as dinitrofluorobenzene, sodium salicylate, sodium benzoate and phenylbutazone suggest that urate and chloride are transported by different mechanisms. 3. Urate influx seems to depend on intracellular glycolysis. The results obtained on red blood cells after glycolysis inhibition agree with those obtained on ghosts where metabolism does not take place. 4. The large drop in urate influxes into erythrocytes in the presence of a glycolysis inhibitor and of a passive ion transport inhibitor seems to argue in favour of a dual urate transport mechanism, one for passive diffusion and the other connected with glycolysis. 5. The drop in the urate influx into ghosts in the absence of ATP suggests that the latter might intervene in urate transport by human red cell membranes.     

Is allantoin in serum and urine a useful indicator of exercise-induced oxidative stress in humans?

To assess whether allantoin levels in serum and urine are influenced by exhaustive and moderate exercise and whether allantoin is a useful indicator of exercise-induced oxidative stress in humans, we made subjects perform exhaustive and moderate (100% and 40% VO₂max) cycling exercise and examined the levels of allantoin, thiobarbituric acid reactive substances (TBARS) and urate in serum and urine. Immediately after exercise at 100% VO₂max, the serum allantoin/urate ratio was significantly elevated compared with the resting levels while the serum urate levels was significantly elevated 30 min after exercise. The serum TBARS levels did not increase significantly compared with the resting levels. Urinary allantoin excretion significantly increased during 60 min of recovery after exercise, however, urinary urate excretion decreased significantly during the same period. The urinary allantoin/urate ratio also rapidly increased during 60 min of recovery after exercise. Urinary TBARS excretion decreased during the first 60 min of the recovery period and thereafter significantly increased during the latter half of the recovery period. On the contrary, after 40% VO₂max of exercise, no significant changes in the levels of urate, allantoin and TBARS in serum or urine were observed. These findings suggest that allantoin levels in serum and urine may reflect the extent of oxidative stress in vivo and that the allantoin which appeared following exercise may have originated not from urate formed as a result of exercise but from urate that previously existed in the body. Furthermore, these findings support the view that allantoin in serum and urine is a more sensitive and reliable indicator of in vivo oxidative stress than lipid peroxidation products measured as TBARS.     

Specificity in the recognition of crystals by antibodies

We show that IgG molecules isolated from the serum of rabbits injected with crystals of monosodium urate monohydrate, magnesium urate octahydrate and allopurinol, can each catalyze the nucleation of the same type of crystal to which they were exposed. These results generalize previous findings related to monosodium urate monohydrate and assess the idea that the invasion of a foreign crystal into an organism may amplify a population of antibodies which bear in their binding sites an imprint of the crystal surface structure. Such antibodies further act as nucleating templates which accelerate crystal formation in vitro. Antibodies from rabbitscan injected with sodium urate crystals do not cross-react or cross-react only to a low extent with antibodies isolated from rabbits injected with crystals of either magnesium urate or allopurinol. These results indicate a high specificity of the elicited antibodies, as these can distinguish between nuclei of crystals having similar molecular and structural characteristics.     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

Urate attenuates oxidation of native low-density lipoprotein by hypochlorite and the subsequent lipoprotein-induced respiratory burst activities of polymorphonuclear leukocytes

Oxidation converts native low-density lipoprotein (LDL) into a signal molecule promoting inflammatory processes during atherogenesis. The exact contribution of different antioxidants in prevention of LDL oxidation is not known. Uric acid efficiently scavenges oxidants including hypochlorite. We investigated the effect of different urate concentrations (25-500 mol/l) on the oxidation of isolated native LDL by sodium hypochlorite (1000 mol/l). While relative electrophoretic mobility declined continuously with increasing urate concentrations in the oxidation medium, lipid peroxidation as measured by TBARS was blunted only at high molar urate/NaOCl ratios. By decreasing oxidative modifications, urate dose-dependently (beginning with a urate/NaOCl ratio of 1:40) diminished stimulatory effects of oxidized LDL on the respiratory burst of resting polymorphonuclear leukocytes (PMNL). Protecting effects of urate against the proinflammatory action of oxidized LDL on activated cells were evident only at a molar urate/NaOCl ratio of 1:2 suggesting different sensitivities of PMNL to LDL oxidation state in dependence on their activity state.     

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cmx75-microm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.     

ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.     

ABCG2 is a High-Capacity Urate Transporter and its Genetic Impairment Increases Serum Uric Acid Levels in Humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3′-azido-3′-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10^?5). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.     

Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Determination of the cross-points of rat liver peroxisomes,peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Allantoin formation and urate and glutathione exchange in human muscle during submaximal exercise.

Seven males performed two exhaustive cycling bouts (EX1 and EX2) at a work-rate of 90% of maximal oxygen uptake, separated by 60 min. During EX1 there was a significant accumulation of urate (from 0.16 +/- 0.02 to 0.27 +/- 0.03 micromol/kg d.w.) and allantoin (from 0.39 +/- 0.05 to 0.69 +/- 0.14 micromol/kg d.w.) in the muscle. An uptake of urate was observed in early recovery from EX1 (0-9 min: 486 +/- 136 micromol; p <.05). There was no exchange of total glutathione or cysteine over the muscle either during or after exercise, and muscle and plasma total glutathione remained unaltered (p <.05). The glycogen levels were lowered by 40% at the onset of EX2, yet the level of oxidative stress in EX1 and EX2 was similar as evidenced by a similar increase in muscle allantoin in both exercise bouts. The data suggest that urate is utilized as antioxidant in human skeletal muscle and that reactive oxygen species are formed in muscle during intense submaximal exercise. No net exchange of glutathione appears to occur over the muscle either at rest, during exercise or in recovery. Moreover, when an exhaustive exercise bout is repeated with lowered glycogen levels, the level of oxidative stress is not different than that of the first bout.     

Uric acid uptake in erythrocytes of beagle and dalmatian dogs

Uric acid uptake by erythrocytes of Beagle and Dalmatian dogs has been measured, using (2-14C) uric acid. Uptake was characterized by a fast and a slow component. Urate uptake was inhibited by certain purine and pyrimidine derivatives and by anion transport inhibitors. It was dependent on intraerythrocyte glycolysis. Temperature only influenced uptake by the slow component (Q10 = 2.6). Urate uptake by the slow component is apparently due to the transport into the erythrocytes by facilitated diffusion (Km = 6.6 mmol/l, Vmax = 390 mumol/l/min), whereas the fast component exhibits an adsorption of urate on erythrocyte surface. No difference of urate uptake by erythrocytes of Beagle and Dalmatian dogs has been observed.     

The case for low dose diuretics in hypertension: comparison of low and conventional doses of cyclopenthiazide.

In a double blind placebo controlled randomised parallel study the antihypertensive activity and adverse biochemical effects of three doses of cyclopenthiazide were evaluated in patients with mild essential hypertension that had been recently diagnosed or was being treated with a single drug. After a four week placebo washout period 53 patients with diastolic blood pressures between 90-110 mm Hg were randomly assigned to 50, 125, or 500 micrograms cyclopenthiazide or matching placebo for an eight week period of treatment. Blood pressure was measured in the patients'' homes by the same observer every two weeks. Serum urea, electrolytes, urate, and creatinine concentrations and 24 hour urinary sodium excretion were monitored every four weeks and serum magnesium concentration and plasma renin activity at the end of the washout and treatment periods. After eight weeks of treatment systolic and diastolic blood pressures were significantly reduced in patients taking 125 and 500 micrograms cyclopenthiazide when compared with those taking placebo. The decrement in serum potassium concentration (0.6 mmol/l) and increase in serum urate concentration 0.06 mmol/l) were greatest with the 500 micrograms dose, the increase in serum urate concentration alone being significant. No change in serum magnesium concentration or 24 hour urinary sodium excretion was noted with any dose of cyclopenthiazide. Only the 500 micrograms dose of cyclopenthiazide significantly increased the mean plasma renin activity (1.8 (95% confidence interval 0.2 to 3.4)-5.4 (3.9 to 6.8) nmol angiotensin I/l/h); the other doses like the placebo had no effect. Cyclopenthiazide 125 micrograms, a dose lower than is currently marketed, produced a similar hypotensive response to 500 micrograms of the drug without upsetting the biochemical profile.     

Purine interconversions in the Australian termite,Nasutitermes walkeri hill

Purine interconversions leading to urate synthesis were studied over 60 min in isolated fat bodies from freshly collected Nasutitermes walkeri using ¹⁴C-hypoxanthine, ¹⁴C-inosine and ¹⁴C-guanosine. All were taken up, inosine the most efficiently at an initial rate of 0.06 ± 0.009 nmol/min/mg protein. The major purines, nucleosides and nucleotides were separated and examined for radioactivity. Based on uptake data, 1.3% of ¹⁴C-hypoxanthine, 0.3% of ¹⁴C-inosine and 37% of ¹⁴C-guanosine were converted to urate while 3% of the ¹⁴C-guanosine taken up was also salvaged to nucleotides. Feeding experiments with allopurinol showed that there was no significant production of urate via guanine, guanosine and adenosine. Incorporation data indicated the presence of the enzymes purine nucleoside phosphorylase, xanthine dehydrogenase, guanase and that neither inosine nor hypoxanthine could be salvaged.     

Ammonium regulation of urate uptake in Chlamydomonas reinhardtii

Urate was taken up at a negligible rate by Chlamydomonas reinhardtii cells grown on ammonium and transferred to media containing urate plus ammonium or urate plus chloral hydrate or cycloheximide. Addition of ammonium to cells actively consuming urate produced a rapid inhibition of urate uptake whereas the intracellular oxidation of urate was unaffected. Methylammonium but not glutamine or glutamate inhibited urate uptake. Addition of l-methionine-dl-sulfoximine to cells actively consuming urate provoked ammonium excretion, which was accompanied by a rapid inhibition of urate uptake. In cells growing on urate and exhibiting noticeable levels of nitrite-reductase activity, nitrite caused a sudden inhibition of urate uptake whereas nitrate required a time to induce nitrate reductase and to exert its inhibitory effect on uptake. The urate-uptake system did not require urate for induction since the urate-uptake capacity appeared in nitrogen-starved cells. From these results it is concluded that, in Chlamydomonas reinhardtii, ammonium inhibits urate uptake and also acts as co-repressor of the uptake system.     

ABCG2 Dysfunction Increases Serum Uric Acid by Decreased Intestinal Urate Excretion

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.