Gas-chromatographic determination of urinary oxalate

We describe a simple, rapid method for determining urinary oxalate: isolation by precipitation with calcium chloride and conversion to dimethyl oxalate, which then is measured by gas chromatography. To each sample, tracer amounts of 14C-labeled oxalic acid are added, to determine the analytical recovery of urinary oxalate. Analytical recovery of [14C]oxalic acid added to urine specimens ranged from 15 to 95% (mean, 80%), and corrected recovery, based on calculation isotope-dilution techniques, ranged from 98 to 100%. The urinary excretion of oxalic acid by 18 normal men, ages 23 to 43 years, ranged between 9 and 23 mg/24h, with a mean value of 16 mg; that by 68 patients with small-bowel bypass ranged from 60 to 210 mg/24 h, with a mean of 127 mg.     

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.     

Rapid enzymatic determination of urinary oxalate

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.     

Simplified urinary oxalate determination using an enzyme electrode

An enzyme electrode for urine oxalate measurement has been produced using acrylamide gel-entrapped oxalate decarboxylase retained over a CO2 sensor. Urine required pre-treatment with EDTA, but oxalate extraction was not necessary and inhibition by phosphate and sulphate was not apparent until after 10-14 days. The linear range was 0-0.2 mmol/l. Analysis of 50 urine specimens diluted 1 in 10 in pH 3.0 glycine buffer showed good correlation with a widely-used colorimetric method (y = 1.101x-0.018, r = 0.955).     

A new technique for urinary oxalate ion determination

The authors propose a technique for the determination of urinary oxalate ions by high performance liquid chromatography, which may be used for clinical and scientific purposes.     

Automated method for the determination of 5'-nucleotidase in serum by continuous flow analysis

The manual method of Persijn & Van der Slik (J. Clin. Chem. Clin. Biochem. 6, 441-446 (1968): 7, 493-497 (1969); 8, 398-402 (1970) for the determination of serum 5'-nucleotidase has been adapted to the Auto-Analyzer II System. In the Auto-Analyzer II, the incubation temperature for the enzyme reactions is 37 degrees C, and the effective sample speed is 30/h, at a sample/wash ratio of 2:1. There is good agreement and correlation between the manual method and the Auto-Analyzer II method (equation of regression line: y = x + 0.6, correlation coefficient = 0.988; the normal range is 2.9-10.5 U/l for both methods). In routine use, within-run and between-day reproducibility are considerably smaller for the Auto-Analyzer II than for the manual method, especially when large numbers of samples are assayed.     

高锰酸钾褪色法测定尿液草酸

过量的ＣａＣｌ２和无水乙醇在尿液ｐＨ为５时，可与草酸形成草酸钙沉淀。用草酸钙平衡过的０．０２ｍｏｌ／Ｌ乙酸及去离子水洗涤沉淀，可以除去沉淀物中的干扰物质。在６０℃酸性环境中，草酸还原高锰酸钾，使其在５２５ｎｍ处吸光度下降。根据吸光度的变化可计算出尿液中草酸含量。本法平均回收率８８．８％。批内、批间ＣＶ分别为１０．４％、１５．５％。本法无需特殊设备，适合一般常规实验室使用。     

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).     

匀相法直接测定血清低密度脂蛋白胆固醇

目的 探讨匀相法直接测定血清低密度脂蛋白胆固醇的价值。方法 用９７０７Ａ ＬＤＬ－ｃ试剂盒及ＢＥＣＫＭＡＮ ＣＸ７自动生化分析仪直接测定血清ＬＤＬ－ｃ含量。 结果（１）批内变异ＣＶ％为．５５２％￣０．８０５％,批间变异ＣＶ％〈１％;（２）线性关系良好,直线范围可达４．９５９ｍｍｏｌ／Ｌ;（３）经与聚乙烯硫酸沉淀（ｐｖｓ）法测定对照３０例,ｒ＝０．９４１,Ｐ〈０．０５,呈显著相关,回归方程为Ｙ＝０．     

Colorimetric determination of urinary oxalate recovered as calcium oxalate. Application of a simple correction factor for incomplete preciptation.

A method is described for determination of oxalate in urine. Prior to the determination the oxalate is first precipitated from the urine as calcium oxalate. Correction for incomplete precipitation can be made by applying one simple correction formula. The extent of this correction has been established by determination of the recovery of added [14-C] oxalate. The determination of the precipitated oxalate was carried out by an automated version of the colorimetric reaction of oxalate with uranium and 4-(2-pyridylazo)-resorcinol as originally described by Neas, R.E. and Guyon, J.C. in 1972 (Anal. Chem. 44, 799-805).     

Evaluation of a high‐performance liquid chromatography method for urinary oxalate determination and investigation regarding the pediatric reference interval of spot urinary oxalate to creatinine ratio for screening of primary hyperoxaluria

BackgroundUrinary oxalate can provide important clues for the screening and monitoring of children with primary hyperoxaluria (PH), which is a potentially life‐threatening condition. However, little effort has been devoted to improve the oxalate assay in recent years. We have proposed a reliable and cost‐effective high‐performance liquid chromatography (HPLC) method for urinary oxalate determination.MethodsUrine specimens were centrifuged after one‐step derivatization, and the supernatants were subjected to HPLC analysis.ResultsThe method was validated with consistent linearity from 0.0625 to 2.0 mmol/L with coefficients of variation ≤7.73%, good recovery, low carryover, satisfactory sample stability, and analytical specificity. The lower limit of quantification and the limit of detection were 0.03130 and 0.0156 mmol/L, respectively. Imprecision values were ≤2.92% and ≤16.6% for externally and internally produced controls, respectively. The pediatric reference interval of spot urinary oxalate to creatinine ratios was established together with its application in screening of PH in patients with renal diseases, revealing its successful deployment in our laboratory.ConclusionsThis reliable HPLC method could serve as a significant tool to determine urinary oxalate levels for screening and monitoring of children with PH in routine clinical laboratories.     

Development of a reference method for determining urinary oxalate by means of isotope dilution—mass spectrometry (ID-MS) and its usefulness in testing existing assays for urinary oxalate

An accurate and reproducible GC-MS reference method for determining urinary oxalate is presented. With forty 24-h urines, comparisons were made between this reference method and four other existing procedures: a GC method, an HPLC method and two enzymatic assays. The results of the first two methods were in accordance with the GC-MS method. The performance of the enzymatic kits was less satisfactory. The use of a GC-MS reference method in evaluating existing and newly developed methods is recommended.     

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.     

Measurement of flow in large veins by the continuous thermodilution method

Simple methods for measuring percutaneously blood flow in veins characterized by a large oscillatory component of flow are lacking. Therefore, a thermodilution technique with a constant infusion rate was used for the measurement of inferior vena cava flow in anesthetized dogs. The accuracy of the method was studied both in an artificial circuit and in in vivo experiments. The thermal catheter was introduced upstream in a flow of water ranging from 50 to 1,200 ml/min produced in an artificial circuit maintained at 37 degrees C. With a volume of cold injectate of 43 ml/20s, the correlation between the values obtained by direct measurement and by thermodilution technique was highly significant (r = 0.993; n = 37) with a slope practically equal to 1.0 (Y = 1.03 X + 2.42). With the thermal catheter introduced through the jugular vein and an electromagnetic flowmeter probe placed around the exposed vessel, volumetric flows were registered in the subhepatic vena cava, infrarenal vena cava, as well as renal and iliac veins of eight dogs. Comparison of the values obtained by the two techniques yielded a regression equation of Y = 0.96 X + 34.31 with a correlation coefficient r = 0.943 (range 50-1,000 ml/min; n = 40). The continuous injection method was as accurate at low flows as at high. Qualitatively, thermodilution curves were comparable to electromagnetic flowmeter curves, reproducing instantaneously the cyclic respiratory variations. The method is thus particularly suitable for use in those veins in which there is a large oscillatory component of flow.     

Sensitivity of the direct oxalate oxidase assay of urinary oxalate improved

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.