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Calcium oxalate crystals are widespread among animals and plants. In land plants, crystals often reach high amounts, up to 80% of dry biomass. They are formed within specific cells, and their accumulation constitutes a normal activity rather than a pathological symptom, as occurs in animals. Despite their ubiquity, our knowledge on the formation and the possible role(s) of these crystals remains limited. We show that the mesophyll crystals of pigweed (Amaranthus hybridus) exhibit diurnal volume changes with a gradual decrease during daytime and a total recovery during the night. Moreover, stable carbon isotope composition indicated that crystals are of nonatmospheric origin. Stomatal closure (under drought conditions or exogenous application of abscisic acid) was accompanied by crystal decomposition and by increased activity of oxalate oxidase that converts oxalate into CO2 Similar results were also observed under drought stress in Dianthus chinensis, Pelargonium peltatum, and Portulacaria afra Moreover, in A. hybridus, despite closed stomata, the leaf metabolic profiles combined with chlorophyll fluorescence measurements indicated active photosynthetic metabolism. In combination, calcium oxalate crystals in leaves can act as a biochemical reservoir that collects nonatmospheric carbon, mainly during the night. During the day, crystal degradation provides subsidiary carbon for photosynthetic assimilation, especially under drought conditions. This new photosynthetic path, with the suggested name "alarm photosynthesis," seems to provide a number of adaptive advantages, such as water economy, limitation of carbon losses to the atmosphere, and a lower risk of photoinhibition, roles that justify its vast presence in plants.

Background. Hyperoxaluria is a major risk factor for kidney stone formation. Although urinary oxalate measurement is part of all basic stone risk assessment, there is no standardized method for this measurement. Methods. Urine samples from 24-h urine collection covering a broad range of oxalate concentrations were aliquoted and sent, in duplicates, to six blinded international laboratories for oxalate, sodium and creatinine measurement. In a second set of experiments, ten pairs of native urine and urine spiked with 10 mg/L of oxalate were sent for oxalate measurement. Three laboratories used a commercially available oxalate oxidase kit, two laboratories used a high-performance liquid chromatography (HPLC)-based method and one laboratory used both methods. Results. Intra-laboratory reliability for oxalate measurement expressed as intraclass correlation coefficient (ICC) varied between 0.808 [95% confidence interval (CI): 0.427–0.948] and 0.998 (95% CI: 0.994–1.000), with lower values for HPLC-based methods. Acidification of urine samples prior to analysis led to significantly higher oxalate concentrations. ICC for inter-laboratory reliability varied between 0.745 (95% CI: 0.468–0.890) and 0.986 (95% CI: 0.967–0.995). Recovery of the 10 mg/L oxalate-spiked samples varied between 8.7 ± 2.3 and 10.7 ± 0.5 mg/L. Overall, HPLC-based methods showed more variability compared to the oxalate oxidase kit-based methods. Conclusions. Significant variability was noted in the quantification of urinary oxalate concentration by different laboratories, which may partially explain the differences of hyperoxaluria prevalence reported in the literature. Our data stress the need for a standardization of the method of oxalate measurement.

Growth inhibition in acid soils due to Al stress affects crop production worldwide. To understand mechanisms in sensitive crops that are affected by Al stress, a proteomic analysis of primary tomato root tissue, grown in Al-amended and non-amended liquid cultures, was performed. DIGE-SDS-MALDI-TOF-TOF analysis of these tissues resulted in the identification of 49 proteins that were differentially accumulated. Dehydroascorbate reductase, glutathione reductase, and catalase enzymes associated with antioxidant activities were induced in Al-treated roots. Induced enzyme proteins associated with detoxification were mitochondrial aldehyde dehydrogenase, catechol oxidase, quinone reductase, and lactoylglutathione lyase. The germin-like (oxalate oxidase) proteins, the malate dehydrogenase, wali7 and heavy-metal associated domain-containing proteins were suppressed. VHA-ATP that encodes for the catalytic subunit A of the vacuolar ATP synthase was induced and two ATPase subunit 1 isoforms were suppressed. Several proteins in the active methyl cycle, including SAMS, quercetin 3-O-methyltransferase and AdoHcyase, were induced by Al stress. Other induced proteins were isovaleryl-CoA dehydrogenase and the GDSL-motif lipase hydrolase family protein. NADPH-dependent flavin reductase and beta-hydroxyacyl-ACP dehydratase were suppressed.

Precise measurement of plasma oxalate is difficult because the concentration in healthy humans is fairly low (1–3 μmol/L) (1)(2). A colorimetric enzymatic assay that uses oxalate oxidase is commonly used for oxalate detection (2)(3). This assay is fairly straightforward for detecting urinary oxalate, which occurs at concentrations in the millimolar range. Plasma oxalate, however, occurs at concentrations in the micromolar range, and signals generated by oxidase degradation are difficult to detect. Our reference laboratory previously used an oxalate oxidase–based assay that required enzyme immobilization on a nylon coil and uses an HPLC with spectrophotometric detection (1). This method has several disadvantages, including limited automation potential, and requires subjective estimation of peak size. We therefore took advantage of the enhanced sensitivity of currently available spectrophotometers (absorbance measured down to a sensitivity of 0.0001) to develop a plasma assay that uses soluble oxalate oxidase (1)(2)(3)(4). The reagents used were as follows: oxalate reagents A and B and the Oxalate Urine Control Elevated, purchased from Trinity Biotech; hydrochloric acid (0.01 and 12 mol/L), sodium hydroxide (10 mol/L), potassium citrate monohydrate (crystalline; formula weight 324.22), EDTA disodium salt dihydrate (crystalline powder; formula weight 372.24), citric acid monohydrate (granular; formula weight 210.14), and sodium nitrite (crystalline; formula weight 69.00) from Fisher; and oxalic acid dihydrate (formula weight 126.07) and Triton from Sigma. Disposable polystyrene semimicro (1.5 mL) cuvettes were purchased through Fisher. Absorbance was measured with the Beckman Coulter DU800 UV/Visible Spectrophotometer. Stock citrate buffer (0.33 mol/L) was prepared by dissolving 30.5 g of potassium citrate, 50 g of citric acid, and 2 g of EDTA disodium salt in 1 L of distilled, deionized H2O, with a mean (SD) resulting pH of 3.3 (0.2). Working citrate buffer (0.066 mol/L) was prepared from …

Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%–89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.

Several genes including oxalate oxidase (Oxo) are up-regulated in Triticum aestivum L. root tips exposed to Al. To better understand the function of Oxo during Al exposure, the protein level and enzyme activity were measured. The data indicate that both Oxo protein and activity are increased proportionally to the level of root growth inhibition (RGI). A high level of Oxo expression may result in excess H(2)O(2) production which could become toxic and induce cell death. However, the timing of H(2)O(2) production (observed after 24 h) indicates that it cannot be the primary cause of cell death first observed after 8 h. Moreover, at Al concentrations resulting in 50% RGI, we did not observe any cell death in the sensitive cultivar while a punctated pattern of death involving small groups of cells was found in the tolerant cultivar. This pattern was maintained for several days in the tolerant cultivar, suggesting the involvement of a cell death mechanism aimed at replacing epidermal cells intoxicated with Al while root growth is maintained. The accelerated epidermal cell turnover may represent a new detoxification mechanism helping to protect deeper cell layers of the meristematic and elongation zone essential for root growth.

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn.Erysiphe] graminis f.sp.hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encodingHvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.

Among many aspects of plant defence responses to pathogenic infection are changes in the composition of the exocellular matrix. To study potential defences in white lupin (Lupinus albus L.), suspension-cultured cells were treated for 24 h with one of three different elicitors : CuCl 2 , and two fungal elicitor preparations from purified cell walls of yeast and Colletotrichum lindemuthianum. Two subsets of exocellular proteins: ionicallybound wall proteins and proteins secreted into culture medium, were isolated, and their patterns compared following electrophoretic separation. Only a few proteins were observed in culture filtrates with dominating bands at 27, 33, and 42 kDa. About 30 proteins were observed in cell wall extracts. Changes in protein intensities evoked by elicitor treatments depended on the type of elicitor used, the age and composition of lupin cell culture, and concentration of applied fungal elicitor. Based on these observations, ten proteins were chosen for N-terminal sequencing, and sequences 5-30 amino acids long for nine proteins were obtained. Three of the major proteins sequenced were identified as acidic exocellular chitinase, polygalacturonase-inhibiting protein, and germin/oxalate oxidase.

Germin gene expression in barley (Hordeum vulgare L.) seedlings responds to developmental and environmental cues. During seed germination, germin mRNA levels were maximal 2 d after the start of imbibition in control seedlings and declined to low levels by 6 d. When seeds were sown in the presence of 200 mM NaCl, germin mRNA levels were also maximal after 2 d, but NaCl treatment, which slowed seedling growth, prolonged germin gene expression for an additional 1 d. In 4-d-old seedlings, germin mRNA levels were highest in roots and higher in the vascular transition region than in shoots. In roots of 6-d-old seedlings, germin gene expression was regulated by salt shock and plant growth regulators. Induced germin mRNA levels were maximal 8 h after treatment with NaCl, salicylate, methyl salicylate, or methyl jasmonate and 4 h after treatment with abscisic acid and indoleacetic acid. Like germin mRNA, dehydrin mRNA levels were maximal 8 h after NaCl treatment. In contrast, peroxidase mRNA levels declined to less than control levels within 30 min of treatment. Germin gene expression is regulated developmentally by salt stress and by treatments with plant hormones. Since germin is an oxalate oxidase, these result imply that oxalate has important roles in plant development and homeostasis.

The inhibitory effect exerted by water stress on acetylene reduction activity (ARA) by nodulated roots of faba beans (Vicia faba L.) was correlated with a 40% decline in the organic acid pool of nodule cytosol. Oxalate concentration was lowered (-55%) whereas a stimulation of the bacteroid oxalate oxidase concomitantly occurred. This enzyme was characterized by an optimal activity at pH 8 but, as in higher plants, exhibited a K m for oxalate of 1.4 mM and an inhibition by substrate excess. Oxalate provided to bacteroid incubations supported C 2 H 2 reduction up to 2.5 mM whereas higher concentrations were strongly inhibitory. In contrast, purified symbiosomes incubated with oxyleghaemoglobin reduced C 2 H 2 in the presence of oxalate concentrations up to 10 mM. The peribacteroid membrane (PBM), in controlling the oxalate flux to the bacteroids avoided the substrate inhibition which would limit its efficiency. Thus, oxalate present in high concentration in faba bean nodules could play a role as complementary substrate for bacteroids slowing down the nitrogen fixation decline induced by water restricted conditions.

The measurement of oxalate in urine and plasma continues to be difficult, particularly in the presence of ascorbate. We have modified and validated a colorimetric assay involving the use of oxalate oxidase (EC 1.2.3.4). Modification of an HPLC spectrophotometric detector improved sensitivity (to as much as 1000-fold that of conventional spectrophotometers) and allowed measurement of oxalate concentrations less than 1 mumol/L. This provided more than enough sensitivity for measurement of normal concentrations of plasma oxalate. We established reference values for oxalate concentrations in urine and plasma, studied sample handling, and established conditions to avoid ascorbate interference in urine and plasma measurements. Mean analytical recovery of [14C]oxalate from plasma to the filtrate was 86 (SD 10)%; recovery of unlabeled oxalate from filtrate was 87 (SD 9)%. Urinary oxalate excretion rates in apparently healthy controls were 0.11-0.46 mmol/24 h. Plasma concentrations in control subjects were 2.5 (SD 0.7) mumol/L, similar to concentrations determined by recent gas chromatographic and isotope dilution methods. Frozen and acidified urine samples showed no interference from ascorbate when excess ascorbate was avoided. Ingestion of 2 g of ascorbate daily did not increase urinary oxalate in healthy control subjects, but during storage ascorbate was converted to oxalate in all conditions tested.

We evaluated the efficacies of five treatment procedures for eliminating ascorbate interference in the enzymatic determination of urinary oxalate. Aliquots of urine samples, containing different amounts of added ascorbate and oxalate, were individually subjected to ferric chloride, sodium nitrite, sodium periodate, charcoal, or ascorbate oxidase treatment to eliminate ascorbate interference. Oxalate contents of the urine samples were then determined by a banana oxalate oxidase-horseradish peroxidase-linked assay with 3-methyl-2-benzothiazolinone hydrazone and 3-(dimethylamino)benzoic acid as chromogens. Only those urine samples treated with ascorbate oxidase or charcoal consistently gave recovery of oxalate close to 100%. Treatment with other reagents, though improving the recovery of oxalate, gave inconsistent results. On the basis of these data, we describe procedures for simply and reliably assaying oxalate by using banana oxalate oxidase.

Methods for determination of urinary oxalate, which use oxalate oxidase from mosses, barley, banana peel, and beet stems, may suffer interference from physiological concentrations of Cl− and NO−3, usually found in urine (1)(2)(3)(4)(5)(6). The usual daily excretion of Cl− and NO−3 are 10–15 g/day and 0.5 g/day, respectively. The potential interference of these anions is removed either by passing the urine through an ion-exchange column or by precipitation of oxalate from urine and its redissolution prior to oxalate assay (1)(7). This pretreatment complicates the procedure; consequently, the sensitivity and reproducibility of the determination often suffer. In our laboratory, we have purified an oxalate oxidase from leaves of 10-day-old seedling plants of grain sorghum (CSH-5), which is insensitive to physiological concentrations of Cl− and NO−3 (8). In the present report, we describe a new method of oxalate determination using grain sorghum leaf enzyme, which does not suffer from Cl− and NO−3 interference. The seeds of grain sorghum ( Sorghum vulgare var CSH-5) were a gift from M/s Nath Seeds Ltd. (Aurangabad, India). The 10-day-old seedling plants were raised from these seeds in the laboratory according to our method published previously (8). The leaves were collected and …

Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX-53. The molecular weight of the enzyme was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH 4.7 by isoelectric focusing. This enzyme contained 1.12 atoms of manganese and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzyme was 9.5 mM for oxalic acid at the optimal pH 4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I–, Br–, semicarbazide, and hydroxylamine.

We describe an automated (ABA-100) enzymic method for determination of urinary oxalate by use of oxalate oxidase (EC 1.2.3.4) isolated from beet stems. The H2O2 produced by the oxidation of oxalate by oxalate oxidase is measured by coupling with oxidation and conjugation of 3-methyl-3-benzothiazolinone hydrazone with N,N-dimethylaniline with catalysis by horseradish peroxidase. The resulting indamine dye is measured spectrophotometrically by the difference in absorption at 500 and 600 nm. Interfering substances are removed by oxidation with acidic ferric chloride and by cation-exchange chromatography. The assay is sensitive to 5 mg of urinary oxalate per liter, the standard curve is linear to 70 mg/L, and the procedure requires less than 3 h for completion. The within-run CV was less than 1.6%, the between-day CV less than 5.6%. The oxalate oxidase method results in a mean and reference interval for oxalate excretion that are comparable with those by isotope dilution, gas-chromatographic, colorimetric, and other enzymic procedures.

In this simple, sensitive, and rapid enzymatic method for the determination of oxalate in urine or plasma, oxalate oxidase (EC 1.2.3.4) prepared from barley seedlings is used to convert oxalate to carbon dioxide and hydrogen peroxide, which is determined photometrically at 600 nm, with use of horseradish peroxidase, by oxidative coupling of 3-methyl-2-benzothiazoline hydrazine with N,N-dimethylaniline. Plasma is pre-treated by ultrafiltration and co-precipitation of oxalate with calcium sulfate and ethanol, urine by dilution and reversed-phase chromatography on C18 columns. Analytical recovery for urine is 99 +/- 2%, for plasma 92 +/- 3%. The normal range for urinary excretion is 0.10 to 0.56 mmol/24 h, and for the concentration in plasma 0.4 to 3.7 mumol/L. There were no significant sex-related differences in urinary excretion or plasma concentration. Our within- and between-assay coefficients of variation were, respectively, less than 3.4% and less than 6.0% for urine, and less than 1.5% and less than 4.3% for plasma.

This new reagent kit for the quantitative measurement of oxalate in urine is a modification of an earlier Sigma oxalate assay procedure (procedure no. 590), a coupled enzyme assay involving oxalate oxidase and horseradish peroxidase. The new analytical procedure includes methods for processing urine specimens to eliminate interference with oxalate color development at 590 nm by ascorbic acid, divalent cations, and other urinary constituents. The reaction is complete in less than 5 min, and results are linearly related to oxalate concentration up to at least 1 mmol/L. Assay sensitivity and within-run and between-run precision were within the limits acceptable for other urinary oxalate procedures. Analytical recovery of added oxalate was close to 100%. This specific, simple, rapid procedure is suitable for routine clinical use.

A novel spectrophotometric enzymic procedure for estimating oxalic acid in urine is described. Oxalate oxidase, prepared from moss species, converts oxalic acid to hydrogen peroxide and carbon dioxide. Hydrogen peroxide is determined enzymatically with horseradish peroxidase, by oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone with N,N-dimethylaniline; the resulting indamine due is determined spectrophotometrically at 595 nm. Interfering substances are removed by adsorption to ion-exchange resins and oxidation with charcoal, thus avoiding oxalate recovery problems accompanying oxalate isolation. The procedure is rapid, sensitive, linear, and precise. Results agreed well with those obtained with a widely used chemical technique.

In this procedure, oxalate oxidase (EC 1.2.3.4) immobilized in a continuous-flow system is used to determine oxalate in urine. The hydrogen peroxide formed from oxalate is detected by use of a color reaction with peroxidase (EC 1.11.1.7), 3-methyl-2-benzothiazoline hydrazine, and N,N-dimethylalanine. However, urine contains an oxalate oxidase inhibitor, which cannot be removed by heating, ion-exchange resins, or cellulose columns. This makes it necessary to precipitate the oxalate before assay. The overall assay system is accurate (oxalate recovery, 95.9%), sensitive (less than or equal to 5 mumol/L), precise (within-batch CV less than 1.25%, between-batch CV less than 5%), and relatively rapid (60 samples per working day). The assay system has better accuracy than an established chemical method and a gas-chromatographic method, and is considerably less arduous than and correlates well (r = 0.94) with a modified chemical method. The reference interval for urinary oxalate excretion is 0.16-0.56 mmol per day (n = 97). Only nonphysiological concentrations of ascorbate interfere with the assay, by increasing the oxalate result in the overall assay, presumably by post-micturition formation of oxalate from ascorbate in the urine samples.

The direct colorimetric method for urinary oxalate has been modified to improve its sensitivity. Oxalate is precipitated overnight with calcium chloride and ethanol, the precipitate is redissolved, and the oxalate is measured by use of oxalate oxidase (EC 1.2.3.4), methylbenzothiazolinone hydrazone, and dimethylaniline. The color developed is more intense, analytical recovery averages 102%, and the overall imprecision is less than 5%. To assess the accuracy of the method, we used a gas-chromatography comparison method and control sera. Interference from ascorbate is negligible. The modified method retains its simplicity and is less expensive.

For this direct colorimetry of urinary oxalate, commercially available oxalate oxidase (EC 1.2.3.4) is used. The urine is first diluted, to diminish the effect of interfering substances. Analytical recovery of oxalate from urines with five different oxalate concentrations (0.4 to 2.0 mmol/L) ranged from 92 to 109% (mean 99%). The within-day and between-day precision (CV) of the method for a wide range of oxalate concentrations averaged better than 10%. There is good correlation (r = 0.977) between this enzymatic method (y) and the chemical method of Hodgkinson and Williams (x) [Clin Chim Acta 36: 127-132, 1972], the regression equation being y = 1.014x + 0.061. Urines with added ascorbate give falsely increased results. The proposed method is inexpensive and simple to perform.