IE42152B1 - Method for determination of oxalic acid - Google Patents

Abstract

1508086 Oxalic acid determination IRISH STONE FOUNDATION 16 Aug 1976 [15 Aug 1975 14 Nov 1975] 33984/76 Heading G1B [Also in Division C3] A method of measuring the concentration of oxalic acid, or of a metabolite capable of conversion to oxalate, in a substance comprises the steps of (a) reacting the substance with oxalate decarboxylase to convert the oxalic acid to formic acid (b) reacting the formic acid with formate dehydrogenase in the presence of NAD+ (nicotinamide-adanine dinucleatide and (c) measuring formate removal so as to determine the amount of formic acid formed in (a) which is proportional to the oxalic acid content of the substance. The method is applicable to urine or serum samples in the form of a citric extract wherein formate removal is measured spectrophotometrically or fluorometrically. Oxalate decarboxylase may be prepared from Collybia velutipes and formate dehydrogenase from Pseudomonos oxalaticus. An assay kit may be provided for oxalic acid estimation.

Description

The invention relates to a method for measuring the amount of oxalic acid in a substance,'Which method has clinical and industrial applications. The invention also includes an assay kit for use in carrying out the method.

Numerous methods have been proposed for the measurement of oxalic acid. The accurate determination of oxalic acid concentration in urine, for example, presents considerable difficulties. The most frequently used methods involve direct precipitation, solvent extraction and isotope dilution or various combinations of these methods and their limitations have been reviewed by Hodgkinson (Clin. Chem. 16 ; 547-557, 1970).

Shimazono and Hayaishi (J Biol. Chem. 227 : 151-159, 1957) purified an enzyme from Collybla velutipes which specifically decarboxylates oxalic acid to formic acid and C02- The enzyme has been employed by a number of workers to measure urinary oxalate by quantifying the C02 released either manometrically or colourimetrically. These methods suffer eitherfrom erratic recoveries of C02 or techniques too cumbersome for routine laboratory use.

The present invention relates to a method for measuring the concentration of oxalic acid, or of a metabolite capable of conversion to oxalate (as hereinafter defined), in a substance, Which comprises the steps of: a) reacting the substance with oxalate decarboxylase to convert the oxalic acid into formic acid; b) reacting the for.r.ic acid with formate dehydrogenase in the presence of NAD+ (nicotinamide-adenine dinucleotide); ahd 23 c) measuring formate removal so as to determine the amount of formic acid formed in reaction a), which amount is proportional to the amount of oxalic acid present in the substance. - 2 421S2 Preferably, formate removal is measured spectrophotometrically or fluorometrically Metabolites capable of conversion to oxalate are produced by natural functions and may yield' oxalate by oxidation e.g. glyoxyl ate, glycolate and glycine. The formate dehydrogenase is preferably purified b from Pseudomonas oxalaticus while the oxalate decarboxylase is prepared from the myceliurn of Collybia velutipes.

The method of the invention is particularly suitable for the measurement of urinary and serum oxalate but may also be used in determining the oxalate content of processed foodstuffs, alcoholic and non-alcoholic beverages such as beer and coffee, processed foods, and biological fluids.

Using oxalate decarboxylase Mayer et al (Clin Chem 9 ; 334 - 339, 1963) found that CO,, generation from small urine volumes was insufficient for accurate oxalate determination and subsequent developments to improve the accuracy of this approach have resulted in elaborate and time consuming techniques.

This is overcome in the present method by coupling NAD+ - requiring formate dehydrogenase with oxalate decarboxylase, giving measurable and reproducible optical density changes on small,(e.g. about 200 pi.) sample volumes, furthermore substantially less oxalate decarboxylase (0.04 U) -is required than by other methods, and the invention readily lends itself to automation.

In aqueous solutions concentrations in the range of 100 ug% can be readily and accurately determined. Results can be obtained on 200 ul • 14 quantities of fresh urine. Where C counting facilities are available the assay is greatly improved by measuring oxalate on a citrate extract of urine and .correcting the result by the standard isotope dilution formula. 2i3 This modification is simple, rapid and consistently gives recoveries between 95 - 103%. It has been observed by other workers that oxalic acid in urine increases on storage. A major advantage of the citrate extract is that oxalate can be stored in this form without change for at least 3 months. - 3 42152 The invention aiso relates to an assay kit for use with the method comprising oxalate decarboxylase, formate dehydrogenase and NAD+ (formate free). Preferably, the kit also includes citrate buffer of pH 3.0, potassium oxalate (pure), potassium phosphate buffer pH 7.0, sodium formate, EDTA (ethyleneb diaminetetraacetic acid), and 14C oxalic acid (high specific activity). Preferably the formate dehydrogenase and oxalate decarboxylase are lyophilized.

Where the kit is for use in measuring serum oxalate it may additionally comprise calcium sulphate (solid, analai*grade) and bromothymol blue (solution), The oxalate decarboxylase may be prepared from the mycelium of Collybia 10 velutipes as described by Shimazono and Hayaishi (0 Biol.Chem. 227 : 151-159, 1957) and purified through the dialysate of their first acetone fraction.

The purification procedure.is as described by Shimazono and Hayaishi but with the following modification. After the initial extraction of the mycelium in citrate buffer, 0.1 M pH 3.2, the citrate extract is sonicated for 5 half minute intervals at 0 - 5°C. The suspension is then centrifuged as described by Shimazono and Hayaishi and the remainder of their procedure adhered to. The lyophilisate is stable for at least 3 months when stored'at -15°C. The enzyme is also commercially available (Sigma Chemical Co. and'Cambrian Chemicals Croydon London) and gives similar results, cO Formate dehydrogenase (FDH) is purified from Pseudomonas oxalaticus based on the method of Hdpner and Knappe (Bergemyer HU, editor, Methoden der enzymatischen Analyse 2, Aufl, Weinheim, 1509-1513, 1970). The growth of Pseudomonas oxalaticus may be carried out essentially as described by Hdpner and Knappe. For example, 100 ml., 0.8% yeast extract (Difccf), in a 250 ml conical flask, containing 0.1 M formate and 0.001 M pyruvate is inoculated from art agar slope and continuously shaken while growth proceeds at 25°C. After 48 hours 5 ml of this inoculum per flask is used to inoculate 15 x 2 litre flasks each containing 600 ml of minimal medium (see Johnson et al - Biochim Biophys Acta 89 : *Analar and *Difco are trade marks 351-353, 1964). These are shaken during growth (48 hours) and pH maintained between 7.5 - 9.0 by addition of 3 M formic acid + 0.03 M pyruvate, via a sterile syringe. Addition of acid is required only during the final 12 hours of growth, a.visible check being kept on pH □ by the presence of a mixed indicator in the medium, 1 ml/flask (sodium cresol red 0.025%+ sodium thymol blue 0.075%). Cells are harvested, washed and stored as described by Hflpner and Knappe and under these conditions 1.5 g wet bacteria/litre of medium are obtained.

The purification of FDH is carried out as follows based on the method of Hflpner and Knappe. ALL steps are carried out at 3°C.

Extraction Procedure. lOg of wet cells are suspended in 0.05 M histidine hydrochloride solution pH 5.0 and sonicated for 6 minutes in half minute intervals maintaining the temperature at 0 - 5°C. After centrifugation for 20 minutes at 25,000 g the precipitate is resuspended in half the original buffer volume and similarly resonicated for a further 6 minutes.

After centrifuging as above both supernatants are combined and the precipiates discarded.

Acid Step. The pH of the combined supernatants is adjusted to 5.2 with 0.25 N HCl. The addition of acid must be made very slowly, otherwise considerable loss of FDH activity will occur. The solution is centrifuged as above and the pH of the supernatant readjusted to 5.6 with 0.5 N KOH.

The Protamine Sulphate Step used at this stage by Hflpner and Knappe is omitted as we observed considerable loss of activity (30%) during this step.

Ammonium Sulphate Step. Anhydrous ammonium sulphate is added, 0.2142 g/ml supernatant and after stirring is allowed to stand for 30 minutes and then centrifuged· as above for 30 minutes. The precipitate is dissolved in 3 - 5 ml, 0.5 M histidine hydrochloride pH 5.6.

Desalting. The enzyme solution is desalted batchwise as described by Neal and Florini. Anal. Biochem. 55, 328 - 330, 1973, using histidine hydrochloride buffer 0.05 M pH 5.6. 2 mg NAD (formate free) is added to the desalted enzyme solution.

Hydroxyl Apatile Step. To each ml of desalted enzyme is added 0.1 ml Bio Gel HT (original suspension ca. 280 mg/m], Bio Rad Laboratories Richmond Cal. U.S.A.) and centrifuged as above for 10 minutes.

The supernatant is lyophilized in suitable aliquots and stored dry at -20°C.

Collybia velutipes or Flammulina velutipes (Strain S) is deposited at The American Type Culture Collection under No. 13547, while Pseudomonas oxalaticus is deposited at Torrey Research Station, Aberdeen, under strain No. 8642.

The spectrophotometric readings are preferably made using a Spectro20 photometer or spectrum-line photometer suitable for precise measurements at 340, 334 or 366 nm. A Zeiss? PMQ 2 Spectrophotometer reading at 340 nm has been found to be.suitable.

*Zeiss is a trade mark - 6 43152 The invention is illustrated with reference to the following examples: EXAMPLE 1 Measurement of oxalate content of an aqueous solution.

In order to determine the lower limit of the method a series of aqueous recoveries were carried out as follows. To 1 cm cuvettes (10 mm x 10 mm) containing citrate buffer (0.5 M pH 3.0) 0.1 ml, and EDTA (0.05M) 0.1 ml, dilutions of oxalate 2.0 ml, were added. The reaction was started by the addition of 10 ul oxalate decarboxylase, 0.4 U, when the unit is defined as that amount which catalyses the formation of 1 nmole of formate/Min. The reaction was allowed to proceed for 30 min. at 37°C.

The enzyme was replaced by distilled HgO in the blanks and all determinations carried out in triplicate. At this stage 0.2 ml of potassium phosphate buffer (2.0 M pH 7.0), freshly prepared, containing formate free NAD+ (2.4 nmoles), was added to all cuvettes, mixed thoroughly and •fl. read on a Zeiss PMQ 2 Spectrophotometer at 340 nm. This was followed by the addition of 20 μΐ of FDH (0.16 U) and maximum optical density (O.D.) recorded. This usually occurred be tween 6-10 min (Fig. 1) depending on the resultant formate concentration.

EXAMPLE 2 2o Measurement of oxalate content of urine.

Oxalate measurements were carried out both on fresh untreated urines and on urine extracts prepared as follows. To a 25 ml aliquot of a 24 hr urine volume, 5 ul of ^C-oxalate was added. Duplicate aliquots (100 pi) were taken for counting in scintillation vials to which 0.4 ml of distilled 25 HgO and 5 ml of tolueme/Triton X-100 scintillation fluid (see Turner Int J Appl Radiat Isot 19 : 557 - 563, 1968), was added and the radioactivity k estimated in a Packard liquid scintillation counter model 3385. The urine sample was then titrated to pH 5.0 with 2 N KOH and stored at -15°C for a *Zeiss, Triton and Packard are trade marks 43152 minimum of 16 hr. The sample was thawed at room temperature, centrifuged at 3,000 rpm for 10 min and the supernatant decanted and allowed to drain.

The precipitate was extracted in 4.0 ml citrate buffer (0.1 M pH 3.0) for 1 hr at approximately 75°C'. The extract was centrifuged for 5 min at 3,000 rpm to remove any suspended debris. After cooling, aliquots were again removed and counted as described above.

Fresh urine 200 μΐ, or urine extracts 100 μΐ were added to cuvettes (10 mm x 10 mm) containing citrate buffer (0.1 M pH 3.0) 0.25 ml, arid EDTA (0.05M) 0.1 ml. Reaction was started by the addition of oxalate decarboxylase 10 μΐ (0.04 U), io and allowed to proceed for 30 min at 37°C. Oxalate recoveries and aqueous standards were similarly prepared and blanks set up as previously described.

At this stage 1.6 ml of phosphate buffer (0.5 M pH 7.0) containing NAD+ * (2.4 nmoles) was added to all cuvettes, mixed thoroughly and read on a Zeiss PMQ 2 Spectrophotometer at 340 nm. This was followed by the addition of 20 μΐ of FDH (0.16 U) and maximum optical density (O.D.) recorded. This usually occurred between 10 - 15 min (Fig. 1) depending on the resultant formate concentration.

Calculations Oxalate excretion mg/24 hr = AE.V x IJ x 0.09 ε.υ f Where v = volume of sample in cuvette V = final volume in cuvette U = 24 hr urine volume e, the extinction coefficient = aE.V v.c Where c = concentration of oxalate standard (vmoles/ml) *Zeiss is a trade mark «»1«2 f, the dilution factor, is based on the standard isotope dilution formula = dpm/100 μΐ citrate extract dpm/100 ul urine aliquot 0.09 = correction factor for umoles to mg of anhydrous oxalic acid.

RESULTS With increasing oxalate concentrations over a range 93.75 - 2250 pg% a linear increase in optical density consistently occurs (see Fig. II).

The results obtained are less than theoretical values calculated based on o an extinction coefficient (ε) of 6.22 cm/umole (340 nm) for NADH. Some NADH and formate oxidase activity was detectable in all of the FDH preparations, consistent with the observations of Hdpner and Knappe. NADH formation in the assay is therefore not stoichiometric with formate removal; it is however strictly proportional to it. The maximum optical density must therefore be read (see Fig. I) and the e calculated for each enzyme batch. The extinction coefficient is identical using oxalate or formate standards and ranged from 4.80 - 5.23.

The above method can also be used to accurately determine the specific 14 activity of C labelled oxalic acid.

EXAMPLE 3 Measurement of oxalate content of serum.

To the serum, 15 ml, 5 ul of ^C-oxalate (50 uCi/ml) is added and the pH adjusted to 10.6 with 2N KOH. Duplicate, 100 ul, aliquots are taken for counting in scintillation vials as described in Example 2. The serum is ultrafiltered in a model 52 cell using a PM - 30 membrane at 25 psi of nitrogen and at a high stirring rate. The first 10 ml of ultrafiltrate is collected and duplicate aliquots (100 ul) taken for counting. The ultrafiltration normally requires 45 min. To each - 9 42152 of two tubes (2.5 X 11.0 cm) ultrafiltrate, 5 ml, is added followed by two f drops of bromothymol blue indicator and titrated to pH 6.0 with 2N acetic acid, the colour being matched with a citrate-phosphate buffer pH 6.0 containing the indicator. A saturated solution of calcium sulphate, 2 ml, was added to each t tube followed by 13.5 ml ethanol and the contents well mixed and put. in a water bath for one hour at 22°C. After centrifugation for 10 min at 3,000 rpm, the supernatant was removed and the tube allowed to drain. The precipitate was dried in an oven for 30 min at approximately 105°C and the precipitate extracted in 1.0 ml of 0.05 M citrate buffer pH 3.0 for 15 min at 75°C. Both one ml extracts were then combined and any suspended debris removed by centrifugation for 5 min. Aliquots, 10 μΐ, were removed from the extract and counted as before.

Oxalate measurement was carried out as follows on serum extract. Serum extract 0.2 ml, was added to cuvettes (width 4 mm, light path 10 mm)containing 0.1 ml EDTA (0.05 M). The reaction was started by the addition of 10 μΐ of oxa15 late decarboxylase (0.04 U) and the reaction allowed to proceed for 30 min at 37°C. The enzyme was replaced by distilled water in the blanks and all determinations carried out in duplicate. Oxalate recoveries and aqueous standards were similarly prepared. At this stage 0.6 ml of phosphate buffer (0.5 M pH 7.2) containing NAD+, formate free, (1.4 umoles) was added and the reaction completed 20 as described in Example 2. - 10 42152 Calculations Oxalate level ug/100 ml serum = ΔΕ χ V v 9,000 ε X v f 5 Where = volume of sample in cuvette V = final volume in cuvette ε = the extinction coefficient = ΔΕ χ V V x c Where c = concentration of oxalate standard (umoles/ml) 10 f, the dilution factor is based on the isotope dilution formula = dpm/100 yl citrate extract dpm/100 yl serum aliquot 9,000 = correction factor for y moles to ug of anhydrous lb oxalic acid per 100 ml serum = 100 x 9,000 _ « 100

Claims (20)

1. A method for measuring the concentration of oxalic acid, or of a metabolite (as hereinbefore defined) capable of conversion to oxalate, in a substance which comprises the steps of: a) reacting the substance .with oxalate decarboxylase to convert the oxalic acid into formic acid; b) reacting the formic acid with formate dehydrogenase in the presence of NAD + (nicotinamide-adenine dinucleotide); and c) measuring formate removal so as to determine the amount of formic acid formed in reaction a), v/hich amount is proportional to the amount of oxalic acid present in the substance.

2. A method according to claim 1, wherein formate removal is measured spectrophotometricaliy or fluorometrieally.

3. A method according to claim 1 or claim 2, wherein the substance is urine in the form of a citric extract.

4. A method according to any of claims 1 to 3, wherein the oxalate decarboxylase is prepared from the mycelium of Collybia velutipes (Strain S). 5. 10. A method according to claim 8 or claim 9, wherein after extraction of the formate dehydrogenase in solution, the pH of the solution is adjusted to pH 5.2 by adding 0.25 N HCl.

5. A method according to claim 4, wherein the oxalate decarboxylase is purified, and the purification procedure includes extracting the mycelium in citrate buffer, sonicating the extract and then centrifuging the suspension formed.

6. A method according to claim 5, wherein sonication.is carried out for a sucession of half minute intervals at a temperature of between 0 and 5°C.

7. A method according to any of claims 1 to 6, wherein the formate dehydrogenase is extracted and purified from Pseudomonas oxalaticus.

8. A method according to claim 7, wherein the. extraction and purification procedure comprises breaking the cells of Pseudomonas oxalaticus and extracting formate dehydrogenase in solution, precipitating out the formate dehydrogenase,

9. A method according to claim 8, wherein the cells are broken by sonication.

10. 12. An assay kit for use with the method claimed in claim 1 or claim 2, comprising oxalate decarboxylase, formate dehydrogenase and NAD + (formate free).

11. Formate dehydrogenase for use in the method claimed in claim 1, when purified by the procedure described in any of claims 8 to 10.

12. - 12 25 redissolving the precipitated formate dehydrogenase in a buffer solution, then desalting the formate dehydrogenase batchwise using a centrifuge, and adding NAD to stabilize the formate dehydrogenase.

13. An assay kit for use with the method claimed in claim 3 for the measurement of oxalic acid in urine or serum, comprising oxalate decarboxylase, formate dehydrogenase, NAD + (formate free), citrate buffer of pH 3.0, potassium oxalate

14. An assay kit as claimed in claim 13 for the measurement of oxalic acid in serum additionally including calcium sulphate and bromothymol blue (solution)

15. An assay kit as claimed in any of claims 12 to 14, wherein the formate 20 dehydrogenase is in a purified form. 15 (pure), potassium phosphate buffer of pH 7.0, sodium formate, EDTA (ethylenediaminetetraacetic acid), and oxalic acid (high specific activity).

16. An assay kit as claimed in claim 15, wherein the formate dehydrogenase is purified by the procedure described in any of claims 8 to 10.

17. An assay kit as claimed in any of claims 12 to 16, wherein the formate dehydrogenase and oxalate decarboxylase are in a lyophilized form. 25

18. An assay kit as claimed in claim 13, wherein the citrate buffer and the potassium phosphate buffer are in a lyophilized form.

19. A method for measuring the concentration of oxalic acid in urine sub stantially as hereinbefore described with reference to Examnle 2.

20. A method for measuring the concentration of oxalic acid in serum sub stantially as hereinbefore described with reference to Example 3.