Critical evaluation of a commercial enzyme kit (Sigma) for determining oxalate concentrations in urine

The Sigma reagent kit for urinary oxalate determination is reportedly simple, rapid, and specific for oxalate. We evaluated the kit and identified a number of shortcomings. Our investigations suggest that the recommended time for chromophore development is too short and should be doubled. Oxalate recovery during the extraction procedure depends strongly on urine pH. For complete extraction, urine should be acidified to pH 1.8-2.4. We also observed positive interference from ascorbate in urine. This interference was substantial, absorbances produced from ascorbate standards being approximately 80% of those obtained from oxalate standards of similar concentration. Our investigations also indicate the presence of a substance on the Sigma adsorbent that is eluted during the extraction procedure and interferes in the color reaction. These interferences represent potentially major sources of imprecision in the assay.     

An enzymatic method for oxalate automated with the Cobas Fara centrifugal analyzer

Measurement of oxalate in urine has been automated for use with the Cobas Fara centrifugal analyzer. No sample pretreatment other than (a critical) pH adjustment is required. Between-run CVs were less than 4%. Results were linearly related to oxalate concentration to 1000 mumol/L. Ascorbic acid ingestion, up to 5 g of ascorbate daily, caused no demonstrable interference with the assay. This practical, automated method for assaying urinary oxalate is substantially faster than other chemical or enzymatic methods.     

An enzymic method for the spectrophotometric determination of oxalic acid.

A simple method is described for the measurement of urinary oxalate. Oxalate decarboxylase is coupled with NAD+ requiring formate dehydrogenase and the result recorded spectrophotometrically. Accurate determination can be carried out either on urine or a citrate extract or urine. Using the citrate extract procedure, the urinary samples can be stored for at least 3 months without any effect on the oxalate content.     

Five treatment procedures evaluated for the elimination of ascorbate interference in the enzymatic determination of urinary oxalate

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.     

Quantification of urinary oxalate by liquid chromatography-tandem mass spectrometry with online weak anion exchange chromatography

BACKGROUND: Urinary oxalate is commonly measured with an enzymatic assay that is specific but requires a manual clean-up step to reduce ascorbic acid interference. We developed a urinary oxalate assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with anion exchange chromatography and simple sample preparation. METHODS: We added calibrator or urine sample (10 microL) to 10 microL of (13)C2 oxalate and 400 microL of water and performed separation on a Waters OASIS WAX column, flow rate 0.6 mL/min, and then elution for 0.3 min with water containing 2 mmol/L ammonium acetate and 1 mL/L formic acid and for 1.0 min with 750 mL/L methanol containing 20 mL/L ammonia. We detected multiple reaction monitoring transitions m/z 88.6 > 60.5 and m/z 90.5 > 61.5 for oxalic acid and 13C2-oxalate, respectively, with a Quattro micro tandem mass spectrometer in electrospray-negative mode. RESULTS: Oxalate and 13C2-oxalate eluted at 1.2 min. Mean recovery was 95%, limit of detection 3.0 micromol/L, lower limit of quantification 100.0 micromol/L, linearity to 2212 micromol/L, imprecision <6%, and bias <3% at 166, 880, and 1720 micromol/L. Oxalate eluted after the main area of ion suppression. Mean response ratios for urine and aqueous samples, enriched at 200 and 1000 micromol/L, were 3.7% and 5.4%, respectively. No interference was observed from other organic acids. Passing and Bablock regression analysis comparing the Trinity Biotech enzymatic reagent set and LC-MS/MS showed LC-MS/MS = 1.06 (enzymatic assay) -21.2, r = 0.964, n = 110. Bland Altman analysis showed general agreement, with a mean bias of -1.9 mumol/L. CONCLUSION: This LC-MS/MS assay is applicable for quantifying urinary oxalate excretion.     

The influence of freezer storage of urine samples on the BONN-Risk-Index for calcium oxalate crystallization.

This study was performed to quantify the effect of a 1-week freezer storage of urine on its calcium oxalate crystallization risk. Calcium oxalate is the most common urinary stone material observed in urolithiasis patients in western and affluent countries. The BONN-Risk-Index of calcium oxalate crystallization risk in human urine is determined from a crystallization experiment performed on untreated native urine samples. We tested the influence of a 1-week freezing on the BONN-Risk-Index value as well as the effect of the sample freezing on the urinary osmolality. In vitro crystallization experiments in 49 native urine samples from stone-forming and non-stone forming individuals were performed in order to determine their calcium oxalate crystallization risk according to the BONN-Risk-Index approach. Comparison of the results derived from original sample investigations with those obtained from the thawed aliquots by statistical evaluation shows that i) no significant deviation from linearity between both results exists and ii) both results are identical by statistical means. This is valid for both, the BONN-Risk-Index and the osmolality data. The differences in the BONN-Risk-Index results of both procedures of BONN-Risk-Index determination, however, exceed the clinically acceptable difference. Thus, determination of the urinary calcium oxalate crystallization risk from thawed urine samples cannot be recommended.     

Modified enzyme-based colorimetric assay of urinary and plasma oxalate with improved sensitivity and no ascorbate interference: reference values and sample handling procedures.

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.     

Effects of urine dilution on quantity, size and aggregation of calcium oxalate crystals induced in vitro by an oxalate load.

Increasing urinary volume is an important tool in the prevention of calcium renal stones. However, the mechanism of how it actually works is only partially understood. This study aimed at assessing how urine dilution affects urinary calcium oxalate crystallization. A total of 16 male idiopathic calcium oxalate (CaOx) stone-formers and 12 normal male subjects were studied and 4 h urine samples were taken twice, under low (undiluted urine) and high hydration conditions (diluted urine). An equal oxalate load (1.3 mmol/L) was added to both types of urine and the crystallization parameters were assessed. In both stone-formers and normal subjects, the crystallization processes were significantly (p<0.05 or less) more marked in the undiluted urine than in the diluted urine in terms of: a) total quantity of calcium oxalate dihydrate (COD) and calcium oxalate monohydrate (COM) crystals; b) total quantity of crystalline aggregates; and c) aggregation index (i.e., ratio between the area occupied by crystalline aggregates and the area occupied by all the crystals present). The comparison between stone-formers and normal subjects showed that the greatest difference was for the size of COD crystals, which were larger in the urine of the stone-formers. A further important finding was an inverse relationship between changes in urinary volume and in the aggregation index (r = -0.53, p = 0.004). In conclusion, urine dilution considerably reduces crystallization phenomena induced in vitro by an oxalate load in both calcium stone-formers and normal subjects.     

Méthode rapide de dosage de l'acide oxalique urinaire par Chromatographie en phase gazeuseRapid method for the determination of urinary oxalic acid by gas liquid chromatography

A rapid method for the determination of urinary oxalic acid by gas-liquid chromatography is described. The procedure involves extraction of oxalate from urine by tetrahydrofuran followed by evaporation to dryness and subsequent diesterification with the boron-trifluoride propanol. The derivative is extracted with hexane and is detected by FID gas chromatography. Malonic acid is used as internal standard. Analytical recovery ranged from 94 to 105%. The coefficient of variation in replicate aliquots over the entire range is less than 6%. The expected range for our method is calculated to be 44 to 577 μmol of oxalate per 24-h urine.     

Testing the predictability of the relative urinary supersaturation from the Bonn-Risk-Index for calcium oxalate stone formation.

When introducing a new parameter, it is necessary to compare the power of the new measure with already established ones. For a new method it is quite difficult to compete with established methods which have already ascertained sets of data over many years. A formal comparison of the new parameter with the actual "gold-standard" method can be a useful approach to reduce that problem. It cannot be expected that a new measure would reflect the "gold-standard" method in a simple proportionality. Therefore, it is important to find out the accuracy of the prediction of one parameter from the other, based on simple, e.g. linear, functions. A number of methods exist to determine the crystallization risk of calcium oxalate salts from urine. The most established method is the calculation of the relative urinary supersaturations with respect to these salts using the EQUIL-program, a program computing the equilibrium concentrations of complexes of primary cations and anions commonly found in urine. The Bonn-Risk-Index (BRI) is a new strategy for the evaluation of the risk of calcium oxalate formation, by performing crystallization experiments on native unprepared urine samples. Although the analytical and computational efforts of both approaches are quite different (relative supersaturation = high, BRI = low), the measurements revealed a considerable and significant linear relationship between the relative urinary calcium oxalate supersaturation, and BRI. We were, therefore, interested in predicting the relative supersaturation from the BRI and in the accuracy of this prediction.     

Quantification of urinary oxalate with oxalate oxidase from beet stems

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.     

Modifications to commercial oxalate oxidase based determination of urinary oxalate: a method suitable for routine clinical analysis

A commercially available assay (Sigma) for urinary oxalates based on the oxalate oxidase/peroxidase catalyzed reaction produced, in our hands, recoveries of added oxalate that varied from patient to patient. A modification of the assay is reported that involves the chromatographic separation of interfering substances, such as ascorbate and uric acid, from oxalate prior to analysis. With the modification recoveries of added oxalate were essentially complete and showed less patient-to-patient variability. Correlation with an alternate procedure was greatly improved.     

The diagnostic value of urinary enzyme measurements in hypertension

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.     

Effect of tetrasodium EDTA on enzymatic determinations of urinary oxalate

We studied the effects of pretreating urine samples with tetrasodium EDTA (TEDTA) before measuring urinary oxalate with an enzymatic kit (Sigma). Mean analytical recovery of added oxalic acid was only 49% (SD +/- 13%) when the assay was performed as recommended by the manufacturer, but treating samples with TEDTA improved recoveries (96 +/- 10%). In 20 unselected 24-h urine samples assayed with and without TEDTA treatment, the mean oxalate concentrations were significantly (P less than 0.001) different: 15.6 +/- 8.7 and 12.2 +/- 7.9 mg/L, respectively. TEDTA-treated urine samples stored for 14 days at -20 degrees C lost 20% of their oxalate concentration. Use of TEDTA simplifies sample preparation by eliminating the alkalinizing step needed to dissolve EDTA or disodium EDTA.     

An inexpensive method for sensitive enzymatic determination of oxalate in urine and plasma

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.     

Inclusion of proteins into calcium oxalate crystals precipitated from human urine: a highly selective phenomenon

The abundance of protein in the matrix of calcium oxalate uroliths has fueled speculation regarding its role in stone genesis. In this study, we wanted to characterize the composition of the proteins associated with early stages of calcium oxalate crystallization in urine. Calcium oxalate crystallization was induced in urine from healthy men and women by the addition of an oxalate load. The crystals were harvested and demineralized, and the proteins remaining were separated and characterized by polyacrylamide gel electrophoresis and Western blotting. Most urinary proteins were not detected in the crystals or were present in only small quantities. The most abundant urinary macromolecule, Tamm-Horsfall glycoprotein, was notably absent from the crystal extracts. The predominant protein associated with the crystals, a previously unknown urinary constituent that we call crystal matrix protein (CMP; molecular mass, 30,000 Da), was more prevalent in the crystals derived from female urine. We conclude that most urinary proteins play no direct role in calcium oxalate crystal formation. However, the protein CMP exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis.     

Oxalate is overestimated in alkaline urines collected during administration of bicarbonate with no specimen pH adjustment

We compared measurements of daily urine oxalate excretion in urines collected at the prevailing urine pH with measurements of urine oxalate excretion in urines collected into 20 mL of 6 mol/L HCl. We studied eight healthy adults fed constant diets. Urines were collected during control conditions and, in each subject, during the administration of NaCl, KCl, NaHCO3, or KHCO3, 90 mmol/day. Daily urine oxalate excretion calculated for collections made in acid averaged 271 (SD 79) mumol/day and did not vary with any of the salt supplements. When urines were collected at ambient urine pH (average 5.94, SD 0.23) during control conditions, and during the administration of NaCl or KCl, urine oxalate excretion averaged 263 (SD 88) mumol/day, a value not different from that for collections in acid. However, when urine was collected with no pH adjustment during NaHCO3 or KHCO3 administration (average pH 6.90, SD 0.14), apparent urine oxalate excretion averaged 398 (SD 132) mumol/day, significantly (P less than 0.025) exceeding the mean observed when urines were collected in acid. Moreover, the percentage increase in apparent oxalate excretion increased with urinary pH. These observations reinforce recommendations that urine specimens for measurement of oxalate be collected in acid to avoid the increase in apparent oxalate content that occurs during collection of alkaline urines. This increase presumably results from the well-known in vitro nonenzymatic conversion of ascorbate to oxalate.     

Gas-chromatographic estimation of urinary oxalate and its comparison with a colorimetric method

We describe a simple, specific gas-chromatographic method for urinary oxalate. Its specificity was evaluated by precipitating the oxalate as its calcium salt from urine, followed by methylation of the oxalate with boron trifluoride/methanol and subsequent gas-chromatographic separation and quantitation of the dimethyl oxalate. [U-14C]Oxalate and n-decanoic acid are used as internal standards. Analytical recoveries ranged from 93.8 to 97.7% for oxalate-supplemented urine. Twenty replicate analyses of urines containing typical concentrations of oxalate gave CVs of 10.0, 9.1, and 8.3% for low, medium, and high concentrations, respectively. Day-to-day precision (CV) for single analyses repeated on 20 days was 8.6% for the low urinary oxalate concentration. The lower limit of detection of urinary oxalate is 25 mumol/L. Results by our method correlated well (r = 0.95) with those by a colorimetric method (Clin. Chim. Acta 36: 127, 1972) but averaged 68.4% of those obtained colorimetrically (n = 75 samples, p less than 0.001). The expected range for our method is calculated to be 80 to 500 mumol of oxalate per 24-h urine (mean +/- 1 SD: 280 +/- 100).     

Studies on some possible biochemical treatments of primary hyperoxaluria.

The effects of some putative inhibitors of oxalate production or urinary oxalate excretion have been investigated in the Cynamolgus monkey and in patients with Type I primary hyperoxaluria (hyperoxaluria with glycollic aciduria). Sodium-1-hydroxybutan-sulphonate, D,L-phenyllactate, succinimide and isocarboxazide did not reduce the urinary oxalate excretion in the monkeys. Pyridoxine reduced the excretion of oxalate and glycollate in some patients, and its therapeutic use has been documented over a five-year period. Succinimide, which has been used by other workers for the treatment of non-hyperoxaluric stone formers, did not decrease the excretion of either oxalate or glycollate in three patients in whom it was tried. It did not change the inhibitory activity of the urine with respect to the growth and aggregation of calcium oxalate crystals in any of the three patients, and it did not have any consistent effect on the excretion of calcium oxalate crystals in the one patient who had detectable crystaluria before treatment. We have identified several metabolites of succinimide in the urine of patients taking the drug. These include 2,3-dehydrosuccinamic, 2-hydroxysuccinamic and 3-hydroxysuccinamic acids. Isocarboxazide, cholestyramine and thiamine did not affect the urinary oxalate excretion in the patients. The significance of these observations from the viewpoint of the treatment of primary hyperoxaluria is discussed.     

Measuring urinary glycosaminoglycans in the presence of protein: an improved screening procedure for mucopolysaccharidoses based on dimethylmethylene blue.

Earlier we described a simple and reliable screening procedure in urine for mucopolysaccharidoses based on the color reaction of glycosaminoglycans (GAGs) with dimethylmethylene blue. At physiological concentrations of urinary protein, we observed an obvious interference by protein in the assay. By modifying the assay, we abolished the protein interference. The modified procedure is not disturbed by human serum albumin, IgG (both tested with as much as 5 g/L of protein), or urinary proteins. The modified procedure appeared as reliable as the original. No false-negative results were found in a series of 26 urine samples from patients with mucopolysaccharidoses (sensitivity 100%). In a series of 405 urine samples offered for metabolic screening, 24 samples with increased GAG content and normal GAG composition were seen (specificity 94%). The method may also be applicable for determining GAG in other body fluids or solutions containing protein.