Determination of oxalate in urine and plasma using reversed-phase ion-pair high-performance liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II)-electrogenerated chemiluminescence detection

Using a gas-chromatographic method, we examined the effects of phosphate concentration, added calcium chloride, and pH on precipitation of oxalate from urine. All three factors are important, but the pH of precipitation is particularly so, especially in the presence of even normal concentrations of ascorbic acid. At pH 8, increases in measured oxalate ranged from 20% at an ascorbic acid concentration of 1 mmol/L to more than 300% at 15 mmol/L. Ascorbic acid is rapidly converted to oxalate at alkaline pH. We also investigated the stability of both untreated and acidified urine containing ascorbic acid during storage for up to one month at -70, -20, and 4 degrees C, and room temperature. After one month, untreated collections were stable at -70 degrees C and acidified collections at -20 and -70 degrees C. We recommend conditions for assay and storage of urine specimens that are to be assayed for oxalate under which positive interference by ascorbic acid is minimized.

Factors affecting measurement of urinary oxalate.

Morphology of Urinary Tract Concretions.- I. Definitions.- II. External Appearance of Urinary Calculi in Relation to Site of Recurrence and Type of Stone.- III. Physical Properties.- 1. Hardness.- 2. Density.- 3. X-Ray Absorption.- IV. Chemical Composition of Uroliths.- 1. Compounds.- 2. Ash and Mass Elements.- 3. Trace Elements.- V. Structural Composition of Uroliths.- 1. Aggregate Structural Description.- a) Central Region.- b) Peripheral Region.- 2. Urolithogenesis as the Basis of Aggregate Configuration.- 3. Urolith Phases.- a) Calcium Oxalate.- b) Uric Acid - Uric Acid Dihydrate.- c) Apatite.- d) Struvite.- e) Ammonium Dihydrogenurate.- f) Brushite.- g) Cystine.- h) Xanthine.- i) Organic Matrix.- VI. Urolith Analysis.- 1. Qualitative Chemical Analysis.- a) Processing of Calculous Material.- b) Analytic Procedure.- c) Discussion.- 2. Quantitative Chemical Analysis.- a) Sample Processing.- b) Analytic Procedure.- c) Discussion.- 3. X-Ray Diffraction.- a) Basic Principle.- b) Sample Processing.- c) Exposure Techniques.- d) Data Evaluation and Comparison of Techniques...- 4. Infra-Red Spectroscopy.- a) Basic Principle.- b) Sample Processing.- c) Measurement Technique and Data Evaluation...- d) Discussion.- 5. Thermoanalysis.- a) Basic Principle.- b) Sample Processing.- c) Data Evaluation.- d) Discussion.- 6. Autoradiography.- 7. Combination Techniques.- 8. Microscopy.- a) Polarizing Microscopy.- b) Transmitted Light Microscopy.- c) Phase Contrast Microscopy.- d) Scanning Electron Microscopy.- e) Combined Microscopic Technique - Laser Micro - spectroanalysis - Polarizing Microscopy - Electron Microprobe.- f) Computer Tomography.- 9. Centralising and Standardising Urolith Analysis.- a) Centralisation.- b) Standardisation.- c) Reporting, Documentation and Data Evaluation.- References.- Epidemiology of Urolithiasis.- I. Historical Overview.- II. The Frequency of Urolithiasis.- III. Type Distribution of Calculi.- 1. Geographic Distribution.- 2. Age and Sex of Stone Patients According to Type of Calculus.- 3. Distribution of Calculi in the Urinary Tract.- IV. Seasonal Variations in Incidence of Recurrent and de novo Stone Disease.- V. Race and Familial Predisposition.- VI. Occupation and Lifestyle.- VII. Constitution, Psychological Factors and Concomitant Disease.- VIII Urinary Calculi in Children.- References.- Pathogenesis of Urolithiasis.- I. Introduction.- II. Theories of Stone-Formation.- 1. Localised Lesions in the Kidney.- a) Randall's Plaques.- b) Anderson's Calcific Droplets.- c) Carr's Pouch (Stewart's Nest).- d) Anderson-Carr-Randall Progression.- e) Intranephronic Calculosis.- 2. Hyperexcretion - Crystallisation Theory.- a) General Principles.- b) Solubility Concept.- c) Saturation of Urine.- d) Crystal Nucleation.- e) Crystal Growth.- f) Crystal Aggregation.- 3. Matrix Theory.- a) General Principles.- b) Crystal Nucleation...- c) Crystal Aggregation.- d) Crystal Adhesion to Cell Walls.- e) Inhibition of Crystal Dissolution.- 4. Inhibitor Theory.- a) General Principles.- b) Inhibitors of Nucleation.- c) Inhibitors of Crystal Growth.- d) Inhibitors of Aggregation.- e) Modifiers of Inhibitory Activity.- f) Naturally Occurring Inhibitors.- g) Synthetic Inhibitors.- 5. Unified Theories.- a) General Principles.- b) "Free Particle" Theories of Stone-Formation...- c) "Fixed Particle" Theory of Stone-Formation.- III. Types of Stone Disease.- 1. Cystine Stone Disease.- a) Introduction.- b) Urinary Risk Factors.- c) Stone Disease.- d) Stone-Formation.- 2. Uric Acid Stone Disease.- a) Introduction.- b) Urinary Risk Factors.- c) Stone Disease.- d) Stone-Formation.- 3. Infected Stone Disease.- a) Introduction.- b) Urinary Risk Factors.- c) Stone Disease.- d) Stone Formation.- 4. Calcium Stone Disease.- a) Introduction... |.- b) Urinary Risk Factors.- c) Stone Disease.- d) Stone-Formation.- 5. Uncommon Stones.- a) Introduction.- References.- Diagnosis of Urinary Calculi.- I. History.- 1. Pain.- a) Incidence.- b) Intensity and Radiation.- 2. Changes in the Urine.- a) Color.- b) Volume.- c) Disorders of Micturition.- 3. Fever.- 4. Predisposing Factors.- a) First Attack or Recurrence.- b) Anatomic Anomalies and Coexisting Disease...- c) Drugs.- d) Individual Dietary History.- II. Examination.- 1. Physical Examination.- 2. Blood Pressure.- III. Imaging Studies.- 1. Ultrasound.- 2. Radiology.- a) Abdominal Plain Film.- b) Intravenous Urography.- c) Retrograde Pyelography.- d) Antegrade Pyelography.- e) Computerized Tomography.- f) Renal Angiography.- g) Micturating Cystogram.- 3. Nuclear Medical Studies.- IV. Laboratory Investigations.- 1. Basal Conditions.- 2. Sample Collection.- 3. Sample Preservation.- 4. Nature and Extent of Investigations.- a) The Basic Screen.- b) Advanced Test Panel.- c) Maximum Test Panel.- References.

Urolithiasis : etiology, diagnosis

Approximate estimates of the ion-acitivity products of calcium phosphate and calcium oxalate in distal tubular urine were derived from the 16-h urinary excretion of calcium, oxalate, citrate, magnesium and phosphate. Urine variables were obtained from 96 normal subjects and 277 calcium stone formers and the calculations were carried out with iterative approximation using the EQUIL2 program. With respect to other ions of importance for the ion-activity products, the urine was assumed to have a fixed composition with pH 6.45. Significantly higher ion-activity products of both calcium phosphate and calcium oxalate were recorded in stone formers. It was concluded that diurnal variations in urine composition and pH might result in peaks of calcium phosphate supersaturation in distal tubular urine whereby a crystallization can occur. In association with abnormalities in terms of promotion and inhibition of calcium salt crystallization, such a precipitation can be of importance for the subsequent formation of calcium renal stones.

Estimated levels of supersaturation with calcium phosphate and calcium oxalate in the distal tubule.

We describe a simple, sensitive assay for oxalate in urine or plasma. Acidified urine is pretreated by dilution with neutral phosphate buffer and passage through a C18 cartridge. Stabilized plasma is diluted with neutral acetate buffer and oxalate extracted using a strong anion exchange cartridge. Treated samples are applied to an ion-paired chromatographic system and oxalate detected electrochemically. Recovery of oxalate from augmented samples exceeded 97% from both urine and plasma. Within- and between-assay coefficients of variation assessed at three concentrations were, respectively, better than 4.1% and 8.4% for urine and 3.9% and 5.2% for plasma. The reference range for urinary oxalate excretion is 109-497 mumols/24 h. The range for plasma oxalate concentration is 0.6-2.8 mumols/L or 0.7-3.9 mumols/L after an overnight fast or without dietary restriction, respectively. Urine and plasma oxalate concentrations from this method, gave correlation coefficients (r) of 0.97 and 0.98, respectively, when compared with those from established oxalate oxidase based assays.

https://journals.sagepub.com/doi/pdf/10.1177/000456329102800607

The determination of oxalate in urine and plasma by high performance liquid chromatography.

Calcium oxalate is an important constituent of the majority of renal stones. Thus measurement of urinary oxalate has become essential in the basic evaluation of the urolithiasis patient. However, the large number of published methods indicate the presence of considerable methodological difficulties1. In this paper we describe our recently developed method for determination of urinary oxalate, based on a reversed-phase ion-pair “high-performance” liquid chromatography (HPLC)2. We also describe a comparison performed between our HPLC method, the colorimetric method of Hodgkinson and Williams3, and a commercial enzymatic method4.

M Asperud

Papers

Determination of urinary oxalate by reversed-phase ion-pair "high-performance" liquid chromatography.