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The authors describe an automatic method of estimation of uric acid based on the colour obtained in the presence of Gaiac resin and peroxidase, and the hydrogen peroxide produced by the action of urate oxidase on uric acid.
Peroxisomes were isolated form derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a "Merkenschlager" cell mill (at 0 degrees C using glass beads). Catalase and urate oxidase, along with low activities of D-amino acid oxidase and L-alpha-hydroxyacid oxidase (glycollate oxidase), were associated with the peroxisomes. No catalase activity was present in glucose repressed cells. When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients. Apparently peroxisomes were destroyed as the cells became fermentative during protoplast preparation. The distribution of glyoxylate cycle enzymes was examined. Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released of present in a labile second class of peroxisomes. Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination. Citrate synthetase was not found associated with a low-density particle as had been previously reported.
INTRODUCTION: Rasburicase is a recombinant urate oxidase that is produced by a genetically modified Saccharomyces cerevisiae and has been approved for prophylaxis and treatment of tumor lysis syndrome in 2001. In several studies, rasburicase, given at a dose of 0.15-0.2 mg/kg for up to 7 d, proved to be highly effective in lowering urate levels. CASE REPORT: We report the case of a patient with chronic lymphatic leukemia (CLL) who experienced tumor lysis syndrome (TLS) with acute renal failure after fludarabine/cyclophosphamide chemotherapy and after bendamustine treatment. During the first episode of TLS, after fludarabine/cyclophosphamide (creatinine 3.3 mg/dL, urate 24.6 mg/dL), the patient received rasburicase 0.2 mg/kg for 3 d. Urate levels decreased below the lower limit of normal and renal function recovered. After bendamustine therapy, given for disease progression 8 months later, TLS with acute oliguric renal failure re-occurred (creatinine 3.1 mg/dL, urate 20.8 mg/dL). The patient was treated with hyperhydration and two doses of rasburicase (0.056 mg/kg), resulting in a prompt decrease of the urate level and recovery of renal function. Both episodes of TLS were successfully treated with rasburicase in a lower dose than recommended by the manufacturer. During a second bendamustine course, TLS was successfully treated by low doses of rasburicase (0.056 mg/kg for 2 d). CONCLUSION: This is the first report of TLS in CLL after bendamustine chemotherapy reported in the literature. Treatment and prevention of TLS by low doses of rasburicase is possible and cost-effective.
Long-term treatment of rodents with peroxisome proliferator chemicals, a group of structurally diverse nongenotoxic carcinogens, leads to liver cancer in a process dependent on the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha). Previous in vitro studies have shown that growth hormone (GH) can inhibit PPARalpha-dependent gene expression by down-regulation of PPARalpha expression and by a novel inhibitory cross-talk involving the GH-activated transcription factor STAT5b. Presently, we evaluate the role of STAT5b in mediating these inhibitory actions of GH on PPAR function using a STATb-deficient mouse model. Protein levels of three PPARalpha-responsive peroxisomal beta-oxidation pathway enzymes (fatty acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and L-bifunctional enzyme) were increased up to two- to threefold in STAT5b(-/-) relative to wild-type control mouse liver, as was the basal expression of two PPARalpha-regulated cytochrome P450 4A proteins. In contrast, protein levels of two PPARalpha-unresponsive peroxisomal enzymes, catalase and urate oxidase, were not affected by the loss of STAT5b. A corresponding increase in expression of fatty acyl-CoA oxidase and L-bifunctional enzyme mRNA, as well as PPARalpha mRNA, was observed in the STAT5b-deficient mice, suggesting a transcriptional mechanism for the observed increases. Although basal liver expression of PPARalpha and its target genes was thus elevated in STAT5b(-/-) mice, the clofibrate-induced level of enzyme expression was unaffected, suggesting that the inhibitory effects of STAT5b are overcome at high concentrations of PPARalpha activators. These findings support the hypothesis that GH and potentially other endogenous activators of STAT5b help to maintain liver PPARalpha function at a low basal level and may thereby moderate PPARalpha-dependent hepatocarcinogenesis and other responses stimulated by exposure to low levels of environmental chemicals of the peroxisome proliferator class.
In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569--571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.
A 14-kDa peroxisomal-matrix protein, named PXP-18, of the yeast Candida tropicalis is a structural and functional homologue of the mammalian nonspecific lipid-transfer protein (identical to sterol carrier protein-2). PXP-18 protected acyl-coenzyme A oxidase (ACO), the rate limiting enzyme of the peroxisomal beta-oxidation of fatty acids, from thermal inactivation at 48 degrees C or 70 degrees C. This effect was dose-dependent and not replaceable either by chicken egg white lysozyme, which is similar to PXP-18 (insofar as it is basic, small, and monomeric), or by bovine serum albumin, a carrier of lipids in the blood. ACO was irreversibly denatured by heat treatment at 70 degrees C for 15 min. However, when ACO and PXP-18 were similarly heat-treated, they formed a large complex at a molar ratio of PXP-18 to ACO subunit that was about one, independent of their initial ratio. This near-stoichiometric complex had ACO activity after a 500-fold dilution and was accompanied by ACO that was free of PXP-18 and indistinguishable from native ACO in size and activity. PXP-18 also protected urate oxidase, another peroxisomal enzyme, from inactivation at 66 degrees C for 15 min and facilitated the renaturation of ACO denatured by 2 M urea. These results indicated that PXP-18 is active in modulating the structure of peroxisomal enzymes in vitro. It is possible that PXP-18 functions as a stress protein or as a part of the system that keeps peroxisomal proteins intact.
Genes that are expressed upon a shift to nitrogen-free medium, an event that initiates gametogenesis, were identified in Chlamydomonas reinhardtii by using the differential display technique. Ten different cDNAs were isolated and shown to have increased levels of their transcripts upon removal of the nitrogen source. The initial kinetics of RNA accumulation allowed an ordering of the genes with respect to the timing of their expression, with individual genes being expressed very early, early, intermediately, or late after induction. For very early genes, significantly increased transcript levels were detected within 30 min. This fast response suggests that gene expression is rapidly activated after removal of the nitrogen source. The accumulation of transcripts from the very early, early, and intermediate genes preceded the appearance of mating competence. Though transcript levels of several very early genes fluctuated during subsequent incubation in nitrogen-free medium, most of them exhibited maxima when the highest level of mating competence was attained. One of these very early genes was shown to encode a urate oxidase type-II enzyme.
Dehydroepiandrosterone (DHEA) is an intermediate product in the synthesis of male and female sex hormones in the adrenal cortex of man. In livers of rats and mice DHEA increases the levels of cytochrome P450 IVA and peroxisomal beta-oxidation enzymes associated with peroxisome proliferation. Prolonged treatment of rats with DHEA induces liver tumors that are more frequent in females arising mainly in the periportal regions of the liver lobule (Metzger et al., Toxicol. Pathol. 23, 591-605, 1995). Because of paucity of information on hepatic zonation of peroxisomal response to DHEA and controversial reports on gender-specific differences of its effects the present study was undertaken using qualitative immunohistochemical and quantitative immunoelectron microscopical techniques in addition to Western blotting. Rats were treated for 24 weeks with 0.6% DHEA supplied with diet. Immunoblot analysis revealed marked induction of peroxisomal beta-oxidation enzymes, which by quantitative analysis was equally strong in male and female animals, whilst catalase and urate-oxidase were not increased. Cytochrome P450 IVA, in contrast, was induced significantly stronger in male than in female rats. Immunohistochemistry confirmed the induction of cytochrome P450 IVA showing a marked lobular gradient in female animals with strong induction in pericentral and almost no induction in periportal regions of the liver lobule. In male animals cytochrome P450 IVA was expressed more uniformly across the liver lobule. A similar sex specific zone-dependent response was observed for peroxisomes. DHEA induced in females a significant zonal gradient with marked peroxisome proliferation and a strong induction of peroxisomal hydratase/dehydrogenase in pericentral hepatocytes and a much smaller response in periportal regions. Livers of male animals, in contrast, showed a uniform peroxisomal proliferation to DHEA with only slight zonal differences. The striking homologies of the induction patterns of cytochrome P450 IVA and the peroxisome proliferation in both sexes support the notion of a functional relationship. In view of the almost exclusive periportal localization of DHEA-induced tumors in female rats in contrast to the pericentral localization of the peroxisomal proliferation shown by this study, it seems likely that other factors in addition to peroxisome proliferation may contribute to the hepatocarcinogenic effect of DHEA.
A preparative method for the isolation of peroxisomes from the liver of normal, untreated rats is described. The peroxisome-enriched "light mitochondrial" fraction is layered on a 30% Nycodenz (5-[N-2,3-dihydroxypropylacetamido]-2,4,6-triiodo-N,N'-bis[2, 3-dihydroxypropyl]isophthalamide) solution containing 1 mM tetrasodium EDTA and then centrifuged in an angular rotor for 1 h at 130,000gavg. Peroxisomes are sedimented to the bottom leaving other organelles at the top of the tube. On the basis of morphological and biochemical studies, it is found that the peroxisomes (marker-enzymes catalase and urate oxidase) obtained in this method are not contaminated with lysosomes (marker-enzyme acid phosphatase) and contained very few mitochondria (marker-enzyme succinate-cytochrome c reductase) and microsomal vesicles (marker-enzyme glucose-6-phosphatase).
We recently encountered a destructive case of tophaceous gout in a 57-year-old patient. Despite perfect therapy compliance, the patient failed in the conventional urate-lowering treatment, accounting for the ongoing urate retention and accumulation with progressive tophaceous bulky disease. Application of an experimental scheme of uricolytical therapy on this patient was able to reduce bulky disease significantly. In modern medicine, potent urate-debulking medication with urate oxidase (uricase) derivatives is at our disposal, and it is a challenge for rheumatologists to install the right strategy including innovative approaches with potent uricolytic therapy on the right patient at the right time.
The effect of di-(2-ethylhexyl)phthalate (DEHP) administration on cyanide-insensitive palmitoyl-CoA oxidizing activity in liver was studied. Two weeks of DEHP treatment increased the activity by one order of magnitude in male Wistar rats. A similar effect was also observed in male and female Sprague-Dawley rats and mice, but not in guinea pigs. When the liver was fractionated by differential centrifugation, the activity was concentrated in the light mitochondrial fraction. On the subfractionation of this fraction by sucrose density gradient centrifugation, the activity was distributed in a pattern similar to that of urate oxidase, but not resembling that of glutamate dehydrogenase. These data suggest that a fatty acyl-CoA oxidizing enzyme system which is located in peroxisomes is induced by the administration of DEHP.
The cores of peroxisomes were purified 670 fold from a rat liver homogenate and the protein in the preparation was examined by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Two bands of protein were detected on 10% polyacrylamide gel, and their molecular weights were calculated to be about 32,000 and 27,000. On treatment of the core fraction with alkali, urate oxidase was solubilized and on 10% polyacrylamide gel this fraction gave a single band of protein with an estimated molecular weight of 32,000. These results suggests that the protein component having a molecular weight of 27,000 is the framework protein of the core of rat liver peroxisomes.
Neoplastic disorders may be complicated by acute renal failure (ARF). Different tumors may cause ARF: solid tumors involving the kidney, solid tumors not of hematological origin and not primarily involving the kidney or, more frequently, rapidly developing hematological tumors. The pathogenesis of ARF is different depending on the type of cancer, but the most frequent clinical feature is the acute tumor lysis syndrome, characterized by hyperuricemia, hyperphosphatemia, hyperkalemia, hypocalcemia and acute, frequently oliguric, ARF. The presence of a neoplastic disorder and associated acute illness may sometimes lead to the presence of immunodysfunction, septic complications and multiple organ dysfunction. In these settings patients develop systemic inflammation and diffuse endothelial damage, related to different mediators. Among these substances, in cancer patients, high circulating levels of uric acid are a common finding. Hyperuricemia is caused by the increase of purine metabolism, which is result of the increased cellular turnover or the aggressive cancer chemotherapy regimens that worsen cell lysis and release of purine metabolites. Even if hyperuricemia is not the first insult to the kidney, its development might represent a concomitant factor aggravating other previous or simultaneous insults. The most efficient therapy for lowering uric acid is rasburicase, a recombinant form of urate oxidase, a nonhuman proteolytic enzyme that oxidizes uric acid to allantoin. It is efficacious in reducing serum uric acid levels with associated diuresis more effectively and much faster than allopurinol, and to correct renal dysfunction more rapidly than allopurinol.
A membrane protein fraction was obtained from rat liver rough microsomes by affinity chromatography on a concanavalin A-Sepharose column and then a chelating-Sepharose column. This protein fraction comprised about 2% of the total membrane proteins of rough microsomes and the ribosome-binding activity of ribosome-stripped rough microsomes was predominantly found in this protein fraction, as determined with a liposome assay system. To identify the essential components responsible for the ribosome binding, two approaches were employed. Trypsin treatment of liposomes reconstituted with this protein fraction resulted in the loss of the ribosome-binding activity in parallel with the loss of a dominant band, estimated Mr 34,000, in SDS-polyacrylamide gels. Next, the direct interaction between the binding sites on the membrane of reconstituted liposomes and 60S ribosomal subunits was investigated by photocrosslinking using sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (SAND). The photocrosslinked complex was formed between 60S ribosomal subunits pretreated with SAND and binding-site proteins on the membrane of the liposomes. Then, after the liposomes were solubilized, the complex was isolated by sucrose gradient centrifugation of the binding mixture. The crosslinked proteins were released from 60S ribosomal subunits by cleavage of of crosslinks with beta-ME and analyzed by SDS-polyacrylamide gel electrophoresis and 125I-autoradiography. The 34-kDa protein (p34) was the predominant component that crosslinked to the 60S ribosomal subunits and was found in proportion to the amount of 60S ribosomal subunits added to the system. The p34 was distinguishable by immunoblot analysis from urate oxidase, which is the 34-kDa protein of peroxisomal cores contaminating rough microsomes. These results suggest that the present p34 is a likely candidate molecule for the ribosome-binding activity of rough microsomes.
Urate oxidase (uricase) was immobilized on carboxymethyl high amylose starch cross-linked 35 (CM-HASCL-35), on aminoethyl high amylose starch cross-linked 35, as well as on commercial supports, CNBr-activated Sepharose and diaminodipropylamine agarose. The N -ethyl-5-phenylisoxazolium-3'-sulphonate (Woodward reagent K) gave a high binding but totally inhibited the enzyme activity. Best results were obtained with CM-HASCL-35 using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide as a coupling agent. The immobilized enzyme retained 88% of its initial limit rate [V (max)(app)=16 EU/mg for immobilized uricase versus V (max)=18 EU/mg for the free enzyme], with an apparent decrease of affinity for urate substrate [K (m)(app)=0.17 mM versus K (m)=0.03 mM for the free enzyme]. The coupling yield was 60% and the modified uricase was found more resistant to proteolysis than the free enzyme. The immobilized uricase retained 25% of its initial activity after 60 min in pancreatic proteolysis medium (pancreatin), whereas the free enzyme retained only 5% of its initial activity. The best immobilization yield was obtained with the polymeric support based on CM-HASCL-35 (53%), which gave better results than commercial supports based on agarose.
Although most eukaryotic genomes lack operons, they contain some physical clusters of genes that are related in function despite being unrelated in sequence. How these clusters are formed during evolution is unknown. The DAL cluster is the largest metabolic gene cluster in yeast and consists of six adjacent genes encoding proteins that enable Saccharomyces cerevisiae to use allantoin as a nitrogen source. We show here that the DAL cluster was assembled, quite recently in evolutionary terms, through a set of genomic rearrangements that happened almost simultaneously. Six of the eight genes involved in allantoin degradation, which were previously scattered around the genome, became relocated to a single subtelomeric site in an ancestor of S. cerevisiae and Saccharomyces castellii. These genomic rearrangements coincided with a biochemical reorganization of the purine degradation pathway, which switched to importing allantoin instead of urate. This change eliminated urate oxidase, one of several oxygen-consuming enzymes that were lost by yeasts that can grow vigorously in anaerobic conditions. The DAL cluster is located in a domain of modified chromatin involving both H2A.Z histone exchange and Hst1-Sum1-mediated histone deacetylation, and it may be a coadapted gene complex formed by epistatic selection.
During growth of Aspergillus nidulans in medium containing ammonium the specific activities of most enzymes involved in catabolism of nitrogen sources are low (ammonium repression). The gdhA10 lesion, which results in loss of nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase activity, has been shown to lead to partial relief of ammonium repression of three amidase enzymes as well as histidase. The areA102 lesion led to altered levels of these enzymes but did not greatly affect ammonium repression. The double mutant areA102,gdhA10 was almost completely insensitive to ammonium repression of two of the amidase enzymes and histidase. This suggests that an interaction between the areA and gdhA genes in determining responses to ammonium occurs. Growth of mycelium in medium containing l-glutamate has been found to result in lowered levels of all four enzymes, and this occurs in strains insensitive to ammonium repression. Very strong repression in all strains occurred during growth in medium containing l-glutamine. Relief of these repressive effects of glutamate and glutamine was blocked by cycloheximide. Glutamate and glutamine had similar effects on the production of extracellular protease activity, and growth on glutamine led to low levels of urate oxidase. In contrast to the above enzymes, nitrate reductase was insensitive to the effects of glutamine and glutamate, even though this enzyme is very sensitive to ammonium repression. Although other possibilities exist, it is suggested that there may be mechanisms of general control of nitrogen-catabolic enzymes other than ammonium repression.
1. The metabolism of palmitate and especially of erucate was studied in hepatocytes isolated from rats fed for 3 weeks a diet containing peanut oil (diet, 1), rapeseed oil (diet 2) and partially hydrogenated marine oil (diet 3). 2. The metabolism of palmitate was not significantly influenced by the diet. The rapeseed oil diet caused 1.4 fold and 1.3 fold increase and marine oil diet 3 fold and 2.2 fold increase in the oxidation and chain-shortening respectively of [14-14C]erucic acid in isolated hepatocytes. 3. Cyanide and antimycin A did not inhibit the chain-shortening of erucate in liver cells of rats fed rapeseed oil and peanut oil. The high capacity of the chain-shortening system in hepatocytes of marine oil-fed rats was partially inhibited. 4. Inhibition of the transfer of fatty acids into the mitochondria by lowering the intracellular carnitine concentration and/or by addition of (+)-decanoyl-carnitine resulted in a very pronounced apparent stimulation of the chain-shortening of erucic acid. It is suggested that the chain-shortening system may be virtually independent of the mitochondria, unless the availability of the extramitochondria NAD+ and/or NADP+ is rate-limiting under conditions of extremely low redox potential of the mitochondria. 5. Feeding marine oil or rapeseed oil to the rats induced a 30% increase in catalase activity, a 25--30% increase in urate oxidase activity and a 50% increase in the total CoA in the liver compared to rats fed peanut oil. 6. It is suggested that the increased metabolism of erucate in hepatocytes of marine oil and rapeseed oil-fed rats may be due to the increase in ther peroxisomal beta-oxidation.