Oxalate Synthesis Research Articles

Production of malachite green oxalate and leucomalachite green reference materials certified for purity

Malachite green oxalate (MG oxalate) and leucomalachite green (LMG) have been prepared and certified as pure reference materials. The purities of MG oxalate and LMG were assessed by high-performance liquid chromatography-diode array detection (HPLC-DAD), nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), Karl Fischer titration, ashing and thermogravimetric analysis (TGA). MG oxalate was purified by supercritical fluid extraction (SFE). Prior to purification, commercial MG oxalate purity was estimated to be about 90%. The main impurities present in SFE-purified MG oxalate were identified and quantified using HPLC-DAD. The main impurities were found to be monode-MG (monodemethylated MG oxalate synthesis impurity), 4-(dimethylamino)benzophenone (4-DMABP), MG-carbinol and LMG. The homogeneity of both reference materials was also determined. Issues associated with the stability of LMG and MG oxalate in solution forced an extensive study investigating different parameters i.e. solvent, acid, analyte concentration and temperature. MG oxalate (100 microg/mL) was found to be stable in acetonitrile containing 1% v/v glacial acetic acid for at least 155 days and LMG (100 microg/mL) was stable in acetonitrile for at least 133 days. The final purity value for MG oxalate was 94.3 +/- 1.4% m/m at the 95% confidence interval (or 67% m/m if MG cation is reported). For LMG, the certified purity was found to be 98.8 +/- 0.8% m/m at the 95% confidence interval.

Fructose metabolism to two carbon acids in HepG2 cells

Fructose intake is a risk factor for calcium oxalate stone disease. It has been hypothesized that fructose metabolism promotes oxalate synthesis leading to the increased stone risk. To assess this relationship we have compared glucose and fructose metabolism in cultured HepG2 cells. Oxalate synthesis was measured by ion chromatography (IC), glyoxylate by HPLC, and other anions including sugar phosphates by IC/MS. The rate of oxalate synthesis with 5mM fructose as the main carbohydrate source, 2.08 ± 0.48 nmoles/mg protein/day was 2.9 times higher than that observed with 5 mM glucose, 0.73 ± 0.14 nmoles/mg protein/day. Dose and time dependent effects were observed. Increased syntheses of glycolate and glyoxylate were not detected, suggesting that these organic acids are not involved in this metabolism. Glyceraldehyde is produced from fructose metabolism but not glucose metabolism. It is possible that the metabolism of this aldehyde results in oxalate synthesis, increasing urinary oxalate excretion and the risk of stone formation.

WS5-5 Experimental Endogenous Oxalate Synthesis from Various Oxalate Precursors

WS5-5 Experimental Endogenous Oxalate Synthesis from Various Oxalate Precursors

Effects of Precursors on Preparation of Pd/α-alumina Catalyst for Synthesis of Dimethyl Oxalate

The influence of two palladium precursors, PdCl2 and Pd(NO3)2, on the preparation of catalyst applied in the CO coupling reaction to synthesize dimethyl oxalate was studied. It is revealed that PdCl2 is much more difficult to decompose than Pd(NO3)2 during calcination, and for catalyst prepared from PdCl2, temperatures as high as 773 K are necessary to obtain surface Pd0, while for catalyst prepared from Pd(NO3)2, the corresponding temperature is 473 K. Furthermore, two palladium distributions across the pellet supports are illustrated. The eggshell palladium distribution resulting from using Pd(NO3)2 as precursor was proven more suitable for the present coupling reaction than the uniform palladium distribution obtained by using PdCl2. The results suggest that Pd(NO3)2 is superior to PdCl2 in preparing catalysts for the CO coupling reaction, and the active components on the support outer face have a more efficient utilization than that on the support inner face, which makes moderate reduction of Pd loadin...

Dietary Oxalate and Calcium Oxalate Nephrolithiasis

Patients with calcium oxalate kidney stones are advised to decrease the consumption of foods that contain oxalate. We hypothesized that a cutback in dietary oxalate would lead to a decrease in the urinary excretion of oxalate and decreased stone recurrence. We tested the hypothesis in an animal model of calcium oxalate nephrolithiasis. Hydroxy-L-proline (5%), a precursor of oxalate found in collagenous foods, was given with rat chow to male Sprague-Dawley rats. After 42 days rats in group 1 continued on hydroxy-L-proline, while those in group 2 were given chow without added hydroxy-L-proline for the next 21 days. Food and water consumption as well as weight were monitored regularly. Once weekly urine was collected and analyzed for creatinine, calcium, oxalate, lactate dehydrogenase, 8-isoprostane and H(2)O(2). Urinary pH and crystalluria were monitored. Rats were sacrificed at 28, 42 and 63 days, respectively. Renal tissue was examined for crystal deposition by light microscopy. Rats receiving hydroxy-L-proline showed hyperoxaluria, calcium oxalate crystalluria and nephrolithiasis, and by day 42 all contained renal calcium oxalate crystal deposits. Urinary excretion of lactate dehydrogenase, 8-isoprostane and H(2)O(2) increased significantly. After hydroxy-L-proline was discontinued in group 2 there was a significant decrease in urinary oxalate, 8-isoprostane and H(2)O(2). Half of the group 2 rats appeared to be crystal-free. Dietary sources of oxalate can induce hyperoxaluria and crystal deposition in the kidneys with associated degradation in renal biology. Eliminating oxalate from the diet decreases not only urinary oxalate, but also calcium oxalate crystal deposits in the kidneys and improves their function.

Oxalate synthesis from hydroxypyruvate in vitamin-B6-deficient rats

We studied the effects of an intravenous hydroxypyruvate load on endogenous oxalogenesis in rats receiving a standard diet or a vitamin-B6-deficient diet. Twelve male Wistar rats were randomized to two groups and were fed either a standard diet or a vitamin-B6-deficient diet for 3 weeks. Then the animals received an intravenous infusion of 100 mg/ml (960.6 micromol/ml) of hydroxypyruvate slowly over 10 min. Urine samples were collected just before hydroxypyruvate infusion and at hourly intervals until 5 h afterward. Urinary oxalate, glycolate, and citrate levels were measured by capillary electrophoresis. Hourly urinary oxalate excretion peaked within 2 h, while urinary glycolate excretion peaked at 1 h, after the hydroxypyruvate load in both control and vitamin-B6-deficient rats. Both urinary oxalate and glycolate excretion were higher in vitamin-B6-deficient rats than in control rats. Infusion of hydroxypyruvate increased the 5-h urinary oxalate and glycolate excretion to 0.68% (6.56 micromol) and 0.53% (5.10 micromol) of the administered dose (mol/mol), respectively, in the control rats, while oxalate and glycolate excretion, respectively, increased to 2.43% (23.36 micromol) and 0.79% (7.59 micromol) of the dose in the vitamin-B6-deficient rats. Urinary citrate excretion was significantly lower at baseline and all other times in the vitamin-B6-deficient rats than in the control rats. In conclusion, a hydroxypyruvate load increased endogenous oxalate synthesis in control rats, and its synthesis was even greater in vitamin-B6-deficient rats. Vitamin B6 deficiency also resulted in significant hypocitraturia.

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus Sclerotinia sclerotiorum

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.

Vitamin B6 deficiency augments endogenous oxalogenesis after intravenous l-hydroxyproline loading in rats

The effects of an intravenous hydroxyproline load on endogenous oxalogenesis were compared in rats fed a standard diet or a vitamin B6-deficient diet. Twelve male Wistar rats were randomized to two groups and were fed either a standard diet (control group) or a vitamin B6-deficient diet for 3 weeks. Then the animals were intravenously administered 100 mg (762.6 micromol)/ml hydroxyproline. In the control group, infusion of hydroxyproline increased the 5-h urinary oxalate and glycolate excretion above baseline to 0.27% (2.02 +/- 1.11 micromol) and 0.32% (2.43 +/- 1.60 micromol) of the administered dose (mol/mol), while it was respectively 2.01% (15.24 +/- 2.13 micromol) and 0.00% (-0.02 +/- 0.19 micromol) of the dose in the vitamin B6-deficient group. Therefore, vitamin B6 deficiency augmented endogenous synthesis of oxalate from hydroxyproline by 7.56-fold (15.24/2.02) compared with that in the control group. Urinary citrate excretion was significantly lower at baseline and all other times in the vitamin B6-deficient group compared with the control group. In conclusions, L-hydroxyproline loading augmented endogenous oxalogenesis in the vitamin B6-deficient group without causing hyperglycolic aciduria, and also led to significant hypocitraturia. These findings suggest that hydroxyproline is not metabolized to oxalate via glycolate, but rather via the 4-hydroxyglutamate to glyoxylate pathway (usually requiring vitamin B6-dependent enzymes) even in the presence of vitamin B6 deficiency.

Hydroxyproline ingestion and urinary oxalate and glycolate excretion

Endogenous synthesis of oxalate is an important contributor to calcium oxalate stone formation and renal impairment associated with primary hyperoxaluria. Although the principal precursor of oxalate is believed to be glyoxylate, pathways in humans resulting in glyoxylate synthesis are not well defined. Hydroxyproline, a component amino acid of collagen, is a potential glyoxylate precursor. We have investigated the contribution of dietary hydroxyproline derived from gelatin to urinary oxalate and glycolate excretion. Responses to the ingestion of 30 g of gelatin or whey protein were compared on controlled oxalate diets. The time course of metabolism of a 10 g gelatin load was determined as well as the response to varying gelatin loads. Urinary glycolate excretion was 5.3-fold higher on the gelatin diet compared to the whey diet and urinary oxalate excretion was 43% higher. Significant changes in plasma hydroxyproline and urinary oxalate and glycolate were observed with 5 and 10 g gelatin loads, but not 1 and 2 g loads. Extrapolation of these results to daily anticipated collagen turnover and hydroxyproline intake suggests that hydroxyproline metabolism contributes 20-50% of glycolate excreted in urine and 5-20% of urinary oxalate derived from endogenous synthesis. Our results also revealed that the kidney absorbs significant quantities of hydroxyproline and glycolate, and their metabolism to oxalate in this tissue warrants further consideration.

Treatment of the primary hyperoxalurias: A new chapter

Despite advances in the enzymology, molecular genetics, and clinical knowledge of the primary hyperoxalurias, few treatments are available. Oxalobacter formigenes is a promising new therapy with potential to induce secretion of oxalate into the intestinal lumen, where it can be degraded by the bacteria.

Primary hyperoxaluria type 1: still challenging!

Primary hyperoxaluria type 1, the most common form of primary hyperoxaluria, is an autosomal recessive disorder caused by a deficiency of the liver-specific enzyme alanine: glyoxylate aminotransferase (AGT). This results in increased synthesis and subsequent urinary excretion of the metabolic end product oxalate and the deposition of insoluble calcium oxalate in the kidney and urinary tract. As glomerular filtration rate (GFR) decreases due to progressive renal involvement, oxalate accumulates and results in systemic oxalosis. Diagnosis is still often delayed. It may be established on the basis of clinical and sonographic findings, urinary oxalate +/- glycolate assessment, DNA analysis and, sometimes, direct AGT activity measurement in liver biopsy tissue. The initiation of conservative measures, based on hydration, citrate and/or phosphate, and pyridoxine, in responsive cases at an early stage to minimize oxalate crystal formation will help to maintain renal function in compliant subjects. Patients with established urolithiasis may benefit from extracorporeal shock-wave lithotripsy and/or JJ stent insertion. Correction of the enzyme defect by liver transplantation should be planned, before systemic oxalosis develops, to optimize outcomes and may be either sequential (biochemical benefit) or simultaneous (immunological benefit) liver-kidney transplantation, depending on facilities and access to cadaveric or living donors. Aggressive dialysis therapies are required to avoid progressive oxalate deposition in established end-stage renal disease (ESRD), and minimization of the time on dialysis will improve both the patient's quality of life and survival.

1543: Effects of Hydroxyproline on Endogenous Oxalate Synthesis in Humans

1543: Effects of Hydroxyproline on Endogenous Oxalate Synthesis in Humans

Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells

Failure to detoxify the intermediary metabolite glyoxylate in human hepatocytes underlies the metabolic pathology of two potentially lethal hereditary calcium oxalate kidney stone diseases, PH (primary hyperoxaluria) types 1 and 2. In order to define more clearly the roles of enzymes involved in the metabolism of glyoxylate, we have established singly, doubly and triply transformed CHO (Chinese-hamster ovary) cell lines, expressing all combinations of normal human AGT (alanine:glyoxylate aminotransferase; the enzyme deficient in PH1), GR/HPR (glyoxylate/hydroxypyruvate reductase; the enzyme deficient in PH2), and GO (glycolate oxidase). We have embarked on the preliminary metabolic analysis of these transformants by studying the indirect toxicity of glycolate as a simple measure of the net intracellular production of glyoxylate. Our results show that glycolate is toxic only to those cells expressing GO and that this toxicity is diminished when AGT and/or GR/HPR are expressed in addition to GO. This finding indicates that we have been able to reconstruct the glycolate-->glyoxylate, glyoxylate-->glycine, and glyoxylate-->glycolate metabolic pathways, catalysed by GO, AGT, and GR/HPR respectively, in cells that do not normally express them. These results are compatible with the findings in PH1 and PH2, in which AGT and GR/HPR deficiencies lead to increased oxalate synthesis, due to the failure to detoxify its immediate precursor glyoxylate. These CHO cell transformants have a potential use as a cell-based bioassay for screening small molecules that stabilize AGT or GR/HPR and might have use in the treatment of PH1 or PH2.

Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats

In India, drumstick ( Moringa oleifera Lam. (Moringaceae)) is commonly used as a phytotherapeutic agent. The effect of oral administration of aqueous and alcoholic extract of Moringa oleifera root-wood on calcium oxalate urolithiasis has been studied in male Wistar albino rats. Ethylene glycol feeding resulted in hyperoxaluria as well as increased renal excretion of calcium and phosphate. Supplementation with aqueous and alcoholic extract of Moringa oleifera root-wood significantly reduced the elevated urinary oxalate, showing a regulatory action on endogenous oxalate synthesis. The increased deposition of stone forming constituents in the kidneys of calculogenic rats was also significantly lowered by curative and preventive treatment using aqueous and alcoholic extracts. The results indicate that the root-wood of Moringa oleifera is endowed with antiurolithiatic activity.

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The K m values and specificity constants ( K cat/ K M) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K M values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K M values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD + as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH ( K M 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.

Study and synthesis of organic precursors for salt treatments developed to protect and strengthen building materials and “frescos”

An attempt to find out possible ways to synthesise sulphates and oxalates, to be utilised for strengthening and consolidating limestone and “frescos” surfaces was made. Particularly organic sulphates were prepared and tested for their reactivity with barium salts, trying to obtain slow sulphates hydrolysis and, so, BaSO 4 precipitation. Different solutions were applied on Opificio delle Pietre Dure's standards using different methodologies and applications times. The characterisation of chemical interactions between carbonaceous bodies and solutions was done by X-ray diffraction (XRPD), Fourier transformed infrared spectroscopy (FT/IR), scanning electron microscopy (SEM-EDS). The synthesis of organic sulphates resulted not difficult and the solutions are very easy to use. More difficult was oxalate synthesis. The application results are promising.

Primary hyperoxaluria: from gene defects to designer drugs?

Primary hyperoxaluria is a name given to a group of hereditary disorders characterized by increased synthesis and excretion of the metabolic end-product oxalate, and deposition of insoluble calcium oxalate (CaOx) in the kidney and urinary tract [1]. Only two of the primary hyperoxalurias have been well characterized—type 1 (PH1, MIM 259900) and type 2 (PH2, MIM260000). PH1 is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT, EC 2.6.1.44), and PH2 is caused by a deficiency of the cytosolic/mitochondrial enzyme glyoxylate/hydroxypyruvate reductase (GR/HPR, EC 1.1.1.26/79) [2]. Although the mutant genes and the dysfunctional enzymes are different in PH1 and PH2, the clinical outcome is very similar (i.e. hyperoxaluria, urolithiasis and/or nephrocalcinosis) (Figure 1). Especially in the case of PH1, renal CaOx deposition is likely to lead to kidney failure, after which the combined effects of increased oxalate synthesis and failure to remove it from the body allow widespread deposition of CaOx throughout the body. In addition to hyperoxaluria, PH1 patients often have hyperglycolic aciduria and PH2 patients hyper-l-glyceric aciduria. However, the calcium salts of glycolate and l-glycerate are soluble and, therefore, do not result in any pathology. Genotype–phenotype correlations

Intra-Familial Clinical Heterogeneity: Absence of Genotype-Phenotype Correlation in Primary Hyperoxaluria Type 1 in Israel

Background/Aims: Primary hyperoxaluria type 1 (PH1) is caused by the deficiency of the liver enzyme alanine:glyoxylate aminotransferase which results in increased synthesis and excretion of oxalate. The clinical manifestations of PH1 are heterogeneous with respect to the age of onset and rate of progression. The aim of this study was to investigate possible relationships between a given genotype, the biochemical profile and the clinical phenotype. Methods: We conducted a study of 56 patients from 22 families with PH1 from Israel. The clinical and biochemical data were compiled and the genotype was determined for each family. Results: The prevalent phenotype was of early onset with progression to end-stage renal disease during the first decade of life. Fifteen PH1-causing mutations were detected in 21 families: 10 were first described in this patient population. Marked intra-familial clinical heterogeneity was noted, meaning that there was no correlation between a given genotype and the phenotype. Conclusions: The clinical course of patients with PH1 is not dictated primarily by its genotype. Other genetic and/or environmental factors play a role in determining the ultimate phenotype.

Characterization of Pd–CeO 2/α-alumina catalyst for synthesis of dimethyl oxalate

Effect of adding CeO 2 to Pd/α-Al 2O 3 as a catalyst promoter was investigated by X-ray diffraction, H 2 chemisorption, and X-ray photoelectron spectroscopy methods. The addition of 0.8% CeO 2 (wt%) greatly increased palladium dispersion on the α-Al 2O 3 support and decreased the particle size of palladium. Meanwhile, the addition of CeO 2, increased the surface concentration of Pd. The in situ FTIR spectroscopy study indicated that CO was adsorbed on 1% Pd–0.8% CeO 2/α-Al 2O 3 in the forms of linear species (band at 2089 cm −1) and bridged species (bands at 1967 and 1908 cm −1), and that the latter form was more stable. Methyl nitrite was very active and methoxylate could be observed from the IR spectra after methyl nitrite passed through the catalyst. Catalytic effects were evaluated in a micro-fixed-bed reactor at atmosphere pressure by using vapor-phase catalytic coupling reaction of carbon monoxide to (COOCH 3) 2 (dimethyl oxalate) as a model reaction.

Oxalate absorption and endogenous oxalate synthesis from ascorbate in calcium oxalate stone formers and non-stone formers

Increased rates of either oxalate absorption or endogenous oxalate synthesis can contribute to hyperoxaluria, a primary risk factor for the formation of calcium oxalate-containing kidney stones. This study involves a comparative assessment of oxalate absorption and endogenous oxalate synthesis in subpopulations of stone formers (SFs) and non-stone formers (NSFs) and an assessment of the effect of ascorbate supplementation on oxalate absorption and endogenous oxalate synthesis. Twenty-nine individuals with a history of calcium oxalate kidney stones (19 men, 10 women) and 19 age-matched NSFs (8 men, 11 women) participated in two 6-day controlled feeding experimental periods: ascorbate-supplement (2 g/d) and no-supplement treatments. An oxalate load consisting of 118 mg of unlabeled oxalate and 18 mg of 13C2 -oxalic acid was administered the morning of day 6 of each experimental period. Mean 13C2 -oxalic acid absorption averaged across the ascorbate and no-supplement treatments was significantly greater in SFs (9.9%) than NSFs (8.0%). SFs also had significantly greater 24-hour post-oxalate load urinary total oxalate and endogenous oxalate levels with both treatments. Twenty-four-hour urinary total oxalate level correlated strongly with both 13C2 -oxalic acid absorption (SFs, r = 0.76; P < 0.01; NSFs, r = 0.62; P < 0.01) and endogenous oxalate synthesis (SFs, r = 0.95; P < 0.01; NSFs, r = 0.92; P < 0.01). SFs are characterized by greater rates of both oxalate absorption and endogenous oxalate synthesis, and both these factors contribute to the hyperoxaluric state. The finding that ascorbate supplementation increased urinary total and endogenous oxalate levels suggested that this practice is a risk factor for individuals predisposed to kidney stones.