Oxalate synthesis, transport and the hyperoxaluric syndromes.

This article reviews the mechanisms involved in the synthesis, absorption, excretion and transport of oxalic acid, and the factors controlling these processes in man. The clinical syndromes associated with hyperoxaluria and recurrent calcium oxalate stone disease are reviewed, including new studies that raise the possibility of a generalized oxalate transport abnormality in some patients with renal stone disease. The important role of oxalate in the determination of calcium oxalate solubility in patients with calcium oxalate stone disease is emphasized and future directions for research in the prevention of recurrent calcium oxalate stone disease are discussed.

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger.

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of Pi and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14CO2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this was oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

Oxalate metabolism in end-stage renal disease: the effect of ascorbic acid and pyridoxine.

Oxalate metabolism was studied in ten patients with end-stage renal disease. No patient with primary hyperoxaluria was included in this study. Five patients were on regular haemodialysis and five patients were on chronic ambulatory peritoneal dialysis (CAPD). Oxalate metabolism was assessed by measurement of plasma oxalate concentration (POx), oxalate metabolic pool size (OxMP), tissue oxalate accumulation rate (TOxA), oxalate production rate (OxPR) and dialysis clearance of oxalate (DCOx). These observations were made on three separate occasions in each of the ten patients: initially when the patients were taking a routine ascorbic acid supplement of 100 mg per day; then after a period of 1 month with no ascorbic acid supplement; and then finally after a further period of 1 month's treatment with pyridoxine 800 mg daily. The values for POx, OxMP and TOxA were significantly increased in all ten patients and in the range observed in some patients with type I primary hyperoxaluria. There was no significant difference between immediate prehaemodialysis POx and the POx in the CAPD patients. The DCOx was very much greater during haemodialysis (mean 85 ml/min) than during CAPD (mean 8 ml/min). The acute fall in POx during haemodialysis was greater than 50% of the immediate pre-haemodialysis concentration. Ascorbic acid in a dose of 100 mg/day had no significant effect on the parameters of oxalate metabolism studied. Pyridoxine in a dose of 800 mg/day produced a significant fall in POx in both haemodialysis and CAPD patients.

Utilization of gluconate by Aspergillus niger. II. Enzymes of degradation pathways and main end products.

Aspergillus niger was grown from conidia on a medium with glucose as the source of carbon and potassium nitrate in non-limiting concentration as the source of nitrogen. After the exhaustion of glucose gluconate was added, this compound representing the almost only carbon source in the culture fluid at this time. Gluconate was used rapidly by the preformed mycelium, the main end-products of its metabolization being mycelial substance (including protein), CO2, and oxalate. In cell-free extracts from gluconate utilizing mycelia 8 enzymes of the Embden-Meyerhof (EM) pathway, 5 enzymes of the tricarboxylic acid (TCA) cycle, and an oxalate forming enzyme, oxaloacetate hydrolase (EC 3.7.1.1) were identified. The addition of fluoroacetate together with gluconate resulted in the accumulation of citrate, and in the inhibition of mycelial growth and of accumulation of oxalate. It is concluded that the EM pathway and the TCA cycle are involved in the formation of mycelial substance, CO2 and oxalate from gluconate. There is good correspondence between the rates of gluconate utilization and of oxalate accumulation which were observed immediately after the addition of gluconate and the in vitro activities of gluconokinase and oxaloacetate hydrolase, respectively, at this time.

Effects of sugars and vitamin B-6 deficiency on oxalate synthesis in rats.

Rats fed diets containing galactose as the source of carbohydrate excreted greater amounts of endogenously formed oxalic acid in their urine, compared to rats fed glucose, fructose or sucrose. Rats fed lactose showed similar but less marked effects. The greatest amounts of urinary and fecal oxalate excretions were observed among rats fed galactose and no vitamin B-6. This group had the lowest body weights after 3 weeks of feeding. Control rats fed galactose or lactose diets weighed less than those fed sucrose, glucose or fructose diets. All rats fed galactose developed cataracts. More [14C]oxalic acid was recovered in the urine and kidneys of control rats injected with D-[U-14C]galactose compared to those injected with D-[U-14C]glucose or D-[U-14C]fructose. Similar results were observed in kidneys of vitamin B-6-deficient rats. The possible mechanisms by which galactose and other sugars may be converted to oxalate are discussed.

In vitro synthesis of oxalic acid has no relevant influence on plasma oxalic acid levels in haemodialysis patients.

Plasma oxalic acid concentrations were measured in 13 chronic haemodialysis patients. The mean plasma oxalic acid concentration was 128.0 +/- 48.6 mumol/l, being approximately 8 times higher than the plasma concentration of 14 volunteers (mean = 16.8 +/- 5.2 mumol/l). Ultrafiltrates obtained in vivo from these patients showed a mean oxalic acid concentration of 138.2 +/- 56.5 mumol/l. Since in vivo ultrafiltrates are free of erythrocytes and plasma enzymes, an in vitro synthesis of oxalic acid from precursors by erythrocytes and plasma enzymes can be excluded. As the oxalic acid concentration of plasma corresponded to that of in vivo ultrafiltrates, it is concluded that any in vitro formation of oxalic acid in haemodialysis patients must be negligibly small, and is irrelevant for the measurement of plasma oxalic acid levels in patients receiving regular haemodialysis.

The effects of vitamin B-6 deficiency and hepatectomy on the synthesis of oxalate from glycolate in the rat.

The interrelationship between vitamin B-6 deficiency, liver and extrahepatic tissues with respect to oxalate biosynthesis from [14C1]glycolate in the rat has been investigated. Separate groups of vitamin B-6 deficient and control rats were subjected to total hepatectomy and the metabolism of injected [14C1]glycolate was followed by measuring the respired 14CO2 and analyzing the urine and blood for radioactive metabolites. Vitamin B-6 deficient and control rats were subjected to sham hepatectomies and used as controls. Vitamin B-6 deficient rats showed elevated urinary oxalate compared to controls. Hepatectomy reduced [14C]oxalate excretion and eliminated the B-6 effect. Respiratory 14CO2 was significantly lowered in hepatectomized rats and vitamin B-6 deficiency enhanced the reduction. A difference in oxalate metabolism between normal and vitamin B-6 deficient rat kidneys and possibly other tissues is indicated. The results suggest that the influence of vitamin B-6 on oxalate metabolism is mediated mainly through the liver and supports earlier observations that liver, which contains two oxalate synthesizing enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, is a primary site for the synthesis of oxalate from glycolate.

Oxalate synthesis from [14C1]glycollate and [14C1]glyoxylate in the hepatectomized rat.

Hepatectomy significantly altered the metabolism of [1-14C]glyoxylate and [1-14C]glycollate in the rat. The production of 14CO2 was reduced by 47% and 77-86%, respectively, indicating the involvement of the liver in the oxidation of both substrates. Unidentified intermediates, assumed to be primarily glycine, serine and ethanolamine, were also reduced by over 50%, as would be expected from the removal of the aminotransferase enzymes through the hepatectomy. The biosynthesis of [14C]oxalate from [1-14C]glycollate was reduced by more than 80% in the hepatectomized rat. This suggests that this oxidation is primarily catalyzed by the liver enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, in the intact rat. The limited formation of [14C]oxalate from [14C1]glycollate observed in the hepatectomized rat is probably catalyzed by lactate dehydrogenase or extrahepatic glycolic acid oxidase. Hepatectomy did not significantly alter the rate of formation of [14C]oxalate from [14C1]glyoxylate. However, since saturating concentrations of glyoxylate could not be used because of the toxicity of this substrate, the involvement of glycollic acid oxidase in this oxidation reaction in the intact rat can not be ruled out. In the hepatectomized rat, lactate dehydrogenase appears to be the enzyme making the major contribution, although other as yet not identified enzymes may be contributing. The increased deposition of oxalate in the tissues, oxalosis, may result from the shift in oxalate synthesis from the liver to the extrahepatic tissues.

Hydroxypyruvate-mediated regulation of oxalate synthesis by lactate dehydrogenase and its relevance to primary hyperoxaluria type II.

Hydroxypyruvate (HP) brought about the decarboxylation of [1-14C] glyoxylate nonenzymically at all pH values considered. The rate of decomposition of glyoxylate increased with each increase in the concentrations of the reactants, the pH, and temperature and on the addition of the cations Fe2+, Mn2+, Mg2+, Zn2+, Co2+, and Cu2+. The addition of HP to a purified preparation of lactate dehydrogenase (LDH) catalyzing the oxidation of [1-14C]glyoxylate to [14C]oxalate in the presence of either NAD or NADH inhibited the production of oxalate. These observations have their implications in L-glyceric aciduria (primary hyperoxaluria type II), a syndrome characterized by the accumulation of HP and recurrent oxalosis. They suggest that the accumulating HP may reduce the contribution of intracellular glyoxylate to the formation of oxalate by competitively inhibiting the liver LDH. The involvement of liver LDH in oxalate synthesis and its postulated induction by HP and NAD in vivo are, therefore, reexamined.

Effect of sodium glycolate and sodium pyruvate on oxalic acid biosynthesizing enzymes in rat liver and kidney.

Sodium glycolate feeding (50 mg/100 g body weight/day) to adult male rats for 7 days resulted in increased activities of glycolate oxidase in liver and lactate dehydrogenase in liver and kidney. However, the activity of glycolate dehydrogenase decreased both in liver and kidney. Treatment of sodium pyruvate (100 mg/100 g body weight/day) to the glycolate-fed rats resulted in lowered liver glycolate oxidase activity, and the glycolate dehydrogenase activity was further decreased as compared to glycolate-fed rats in both age groups. However, lactate dehydrogenase activity was not affected by pyruvate feeding in comparison to the glycolate-treated group. It is concluded that glycolate-induced oxalate biosynthesis in rats involves increased activity of liver glycolate oxidase, and pyruvate feeding inhibits glycolate oxidase, thereby decreasing oxalate biosynthesis.

Production of oxalic acid by some fungi infected tubers.

Oxalic acid (as oxalate) was detected in four tubers commonly used for food in Nigeria-Dioscorea rotundata (White yam), Solanum tuberosum (Irish potato), Ipomoea batatas (Sweet potato), and Manihot esculenta (cassava). Whereas healthy I. batata had the highest oxalic acid content, healthy M. esculenta contained the lowest. When all tubers were artifically inoculated with four fungi-Penicillium oxalicum CURIE and THOM, Aspergillus niger VAN TIEGH, A. flavus and A. tamarii KITA, there was an increase in oxalate content/g of tuber tissue. The greatest amount of oxalate was produced by P. oxalicum in D. rotundata tuber. Consistently higher amounts of oxalate were produced by the four fungi in infected sweet potato tuber than in any other tuber and consistently lower amounts of oxalate were produced by the four fungi in Irish potato tuber. Differences in the carbohydrate type present in the tubers and in the biosynthesis pathway are thought to be responsible for variation in the production of oxalate in the different tubers by the four fungi used.

Factors affecting endogenous oxalate synthesis and its excretion in feces and urine in rats.

It has been observed that the feces as well as urine of rats fed diets supplemented with 3% glycine and 5.2% hydroxyproline contain unexpectedly high amounts of endogenously formed oxalate. That intestinal microorganisms do not synthesize significant amounts of oxalate was indicated by the findings that oral tetracycline had no effect on oxalate excretion and that germ-free rats excreted more oxalate than conventional rats. Since little intraperitoneally injected [14C] oxalate appeared in the feces, and rat intestinal mucosa homogenates were found to produce oxalate from a variety of precursors of which glyoxylic acid was far the most important, it is probable that the intestinal mucosa may be an important source of fecal oxalate observed in these studies. Ninety percent of weanling rats fed complete diets supplemented with glycine and hydroxyproline developed urinary stones in 38 days. It has been concluded that in the treatment of patients with histories of calcium oxalate urolithiasis, more concern than is commonly shown should be directed towards the feeding of diets high in precursors of endogenous oxalate synthesis.

Oxalate metabolism and renal calculi.

Changes in oxalate excretion (together with changes in urinary volume) constitute the most important factors in altering the probability of renal stone formation. However, investigations on oxalate metabolism have been sparse, perhaps because of the lack of an accurate method for measuring oxalate in biologic fluids. Available data clearly implicate increased urinary oxalate excretion as the etiological factor in stone formation in two groups of patients--those with primary hyperoxaluria and those with gastrointestinal malabsorption. Evidence for the existence of hyperoxaluria in the patient with the "garden" variety of calcium oxalate stones is less persuasive.

Vitamin B6 deficiency as related to oxalate-synthesizing enzymes in growing rats.

Chronic vitamin B6 deficiency in male rats, 1, 2 and 3 months of age, led to increases in the activities of liver glycolate oxidase and kidney glycolate dehydrogenase as compared to pair-fed controls. Lactate dehydrogenase activity either decreased or showed no change in all three age groups. It is postulated that hyperoxaluria observed in vitamin B6 deficiency is due to two different pathways operative in the liver and kidney separately. A general increase seen in the enzyme activities of livers and kidneys of B6-deficient and pair-fed rats was age related.

Effects of hydroxyproline and vitamin B-6 on oxalate synthesis in rats.

Relationships among dietary hydroxyproline (HP), vitamin B-6 and endogenous oxalate formation have been studied. In the absence of HP, urinary oxalate excretion was greatest among rats fed vitamin B-6-deficient diets. Supplementation of rat diets with 5.2% HP markedly increased the oxalate excretion of rats fed 0, 0.2 or 10 mg of vitamin B-6 per 100 g of diet, the increases being 2-, 19- and 15-fold respectively. The metabolism of several 14C-labeled oxalate precursors was altered in vitamin B-6-deficient rats. The feeding of HP and different levels of vitamin B-6 also altered their metabolism. The feeding of HP to vitamin B-6-deficient rats resulted in a decrease in the amount of 14C-oxalate formed from injected 14C-labeled glycine, glycolate or glyoxylate. In contrast, HP feeding to rats given 0.2 mg of vitamin B-6 per 100 g, resulted in a marked increase in oxalate formation from injected 14C-glycolate, as well as a decrease in respiratory 14CO2 from injected 14C-labeled glycolate and glyoxylate. HP feedings did not significantly alter the metabolism of these two injected compounds to oxalate or CO2 among rats fed the higher level of vitamin B-6, although some elevation of oxalate formation from glycolate was noted. HP feeding reduced the growth rates of all the rats, but growth depression was greatest in the vitamin B-6-deficient group.

Urinary oxalate excretion after large intakes of ascorbic acid in man.

The influence of high dose intake of ascorbic acid on the urinary excretion of oxalate was investigated in five healthy male volunteers. Oxalate was measured by a newly developed specific method using isotachophoresis. With intakes of 10 g ascorbic acid (5 X 2 g daily for 5 days; four subjects) mean urinary oxalate excretion was enhanced from about 50 mg to 87 mg (range 60 to 126 mg) per day. At least 25% of the ascorbic acid was absorbed and excreted with the urine. On discontinuing ascorbic acid administration, oxalate excretion returned to baseline values within 24 h. The time-course of oxalate excretion revealed that following the 3rd dose of 2 g ascorbic acid a plateau in urinary oxalate excretion was reached (0.6 microgram ml-1 min-1) which was not exceeded despite additional 2-g doses of ascorbic acid. On termination of ascorbic acid administration the oxalate excretion rate remained at this level for a further 6 h and then decreased to prestudy rates. No effect of high-dose ascorbic acid ingestion was found on the daily urinary excretion of creatinine, uric acid, and inorganic phosphate. Calcium excretion was slightly reduced. In comparison to the large amounts of ascorbic acid ingested, the increase in urinary oxalate excretion as measured by isotachophoresis in these healthy male volunteers was very low, and is thus similar to the change in urinary content of oxalate which results from consuming normal diets.

In-vivo studies on C2 organic acids in the tissues of rats injected with xylitol and glucose.

Oxalic, glyoxylic, and glycollic acid were determined in rat liver and kidney after injection with [U-14C]-xylitol or [U-14C]-glucose. Neither glucose nor xylitol led to the formation of oxalic and glyoxylic acid, yet glycollic acid was found in both tissues after injection with xylitol. Possible pathways leading from xylitol to glycollic acid are discussed.

The production of (14C) oxalate during the metabolism of (14C) carbohydrates in isolated rat hepatocytes.

Oxalate (14C) was produced during the metabolism of (U-14C) carbohydrates in hepatocytes isolated from normal rats. At 10 mM, the order of oxalate production was fructose > glycerol > xylitol > sorbitol greater than or equal to glucose in the ratio 10 : 4 : 3 : 1 : 1. This difference between oxalate production from fructose and glucose was reflected in their rates of utilisation, glucose being poorly metabolised in hepatocytes from fasted rats. Fructose was rapidly metabolised, producing glucose, lactate and pyruvate as the major metabolites. Glycerol, xylitol and sorbitol were metabolised at half the rate of fructose, the major metabolites being glucose, lactate and glycerophosphate. The marked similarity in the pattern of intermediary metabolites produced by these polyols was not, however, reflected in the rates of oxalate production. Hepatic polyol metabolism resulted in high levels of cytosolic NADH, as indicated by elevated lactate : pyruvate and glycerophosphate : dihydroxyacetone phosphate ratios. The artificial electron acceptor, phenazine methosulphate (PMS) stimulated oxalate production from the polyols, particularly xylitol. In the presence of PMS, the order of oxalate production was fructose greater than or equal to xylitol > glycerol > sorbitol in the ratio 10 : 10 : 6 : 2. The production of glucose, lactate and pyruvate from the polyols was also stimulated by PMS, whereas the general metabolism of fructose, including oxalate production, was little affected. Oxalate (14C) was produced from (1-14C), (2-14C) and (6-14C) but not (3,4-14C) glucose in hepatocytes isolated from non-fasted, pyridoxine-deficient rats. Whilst this labelling pattern is consistent with oxalate being produced by a number of pathways, it is suggested that metabolism via hydroxypyruvate is a major route for oxalate production from various carbohydrates, with perhaps the exception of xylitol, which appears to have an alternative mechanism for oxalate production. The observation that carbohydrates, particularly fructose, contribute to endogenous oxalate production lends support to the hypothesis that a high sucrose consumption contributes to the formation of renal oxalate stones in man.

[Change in ratio between citric and oxalic acid in Aspergillus niger upon exposure to mutagenic factors]. / Izmenenie sootnosheniia limonnoi i shchavelevoi kislot u Aspergillus niger pod vliianiem mutagennykh faktorov.

Stepwise selection of Aspergillus niger producing citric acid in highly buffered molassa media was carried on in order to decrease the biosynthesis of oxalic and gluconic acids using the following mutagenic factors: diethyl sulfate, thiophosphamide, cyclophosphan, N-methyl-N'-nitro-N-nitrosoguanidine (NG), 5-fluorouracil, 1,4-bis-diazoacetylbutane, and UV. The ratio between the acids changed in mutants obtained upon the combined treatment with UV and NG. Mutants producing twice as less oxalic acid were characterized by weak growth and formation of conidia.