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Clinica Chimica Acta, 70 (1976) 71-77 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 7714

DETERMINATION OF OXALIC ACID IN URINE BY ATOMIC ABSORPTION SPECTROPHOTOMETRY

C. KOEHL and J. ABECASSIS Znstitut de Chimie Biologique, Faculte’ de Mddecine, 11, rue Humann, 67085 Strasbourg Ce’dex (France) (Received December 4,1975)

Summary A method is described for determination of oxalic acid in urine using atomic absorption spectrophotometry. The concentration of urinary oxalic acid is calculated by using the difference between two determinations of calcium: first, the excess of calcium in the supernatant after precipitation as calcium oxalate at pH 5, and second, the total calcium determined at pH < 1 (endogenous and added). Analytical parameters (pH of precipitation, temperature, calcium and oxalate added, precipitation time, interfering substances) were studied with the aid of [ 14C]oxalic acid. A constant recovery of 95% of total oxalic acid allows the use of a correction factor. The accuracy and reproducibility of the method make it useful for routine determination of urinary oxalic acid.

Introduction Quantitative determination of oxalic acid in urine can be made by numerous analytical procedures (For review see ref. 1). Most of these methods are time consuming or are not well suited to routine work. All methods using atomic absorption spectrophotometry require precipitation of oxalic acid by a calcium salt. The procedure to estimate calcium in the precipitate, first described by Fraser and Campbell [2] and later modified by Henry [ 31 demonstrates the disadvantage of delicate washing of the precipitate. In contrast with this method, Menache [4] measured the amou;it of calcium remaining in solution, but the results seemed to us still not very precise. We propose here a simple and reliable technique adapted from the method of Menache [4]. The principal factors influencing the recovery of oxalic acid in urine have been optimised after study using [ 14C]oxalic acid.

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Materials and methods Insfrumen tation We used a Perkin-Elmer atomic absorption spectrophotometer model 306 equipped with a Bolling-type burner. The instrument was kept under an aspirating hood. The calcium determination was performed in an ~r/acetylene flame (acetylene was of a purity suitable for medical analysis). The P-emission of i4C-labelled oxalic acid was measured in a liquid scintillation counter Intertechnique model SL 30. Reagents 1. Concentrated hydrochloric acid. 2. Concentrated ~monium hydroxide. 3. overloading oxalic acid solution, 4 g oxalic acid/l. Dissolve 5.95 g of sodium oxalate in 1000 ml of distilled water. 4. Overloading calcium chloride solution, 8 g calcium/l. Dissolve 29.34 g of CaC12(2Hz0) in 1000 ml of distilled water. 5. Lanthanum chloride diluent, 5 g/l. Dissolve 5 g of lanthanum chloride in 1000 ml of distilled water. 6. Solution of “C-labelled oxalic acid, 0.20 ,ugfml, specific activity 0.125 E.tCifml. Reconstitu~ 250 &i of oxalic acid (Radiochemic~ Centre Ltd., Amersham, Buckinghamshire, England) with 2000 ml of distilled water. 7. Liquid scintillator, Unisolve I (Koch Light Laboratories Ltd., ColnbrookBucks, England). 8. Calcium standard solution, 400 mg/l. Add 1 g of anhydrous calcium carbonate to about 200 ml distilled water. Add 6 ml 0.5 M HzS04, and bring the total volume to 1000 ml with distilled water. 9. Overloading phosphate solution, 53.25 g of phosphorus/l. Dissolve 306.05 g of Na~HPO~(2H~O) in 1000 ml of distilled water. 10. Overloading ammonium solution, 58.42 g of ammonia nitrogen/l. Dissolve 223.24 g of ammonium chloride in 1000 ml of distilled water. 11. Overloading magnesium solution, 16 g of magnesium/l. Dissolve 133.76 g of MgC12(6Hz0) in 1000 ml of distilled water. 12. Overloading uric acid solution, 21.32 g of uric acid/l. Dissolve 21.32 g of uric acid in 1000 ml of distilled water. standard procedure The urine specimen was maintained at an acidic pH throughout the entire collection period by the preaddition of 10 ml concentrated WC1 to the collection container. The urine was kept at 4°C until the oxalic acid determination was performed, at which time the urine was adjusted to pH < 1 with concentrated HCl. For each urine specimen, take two 10 ml graduated tubes marked “reference” and “test”. Add 8.0 ml of acidified urine to the two tubes. To both, add 0.4 ml of overloading oxalic acid solution (4 g/l) and 0.4 ml of overloading calcium solution (8 g/l). With a pH meter, adjust the content of the tube marked “test” with NH40H to pH = 5.0. Bring the total volume to 10 ml with distilled water. Allow the contents of the two tubes to precipitate for 48 h at 4°C. _

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Centrifuge at 3000 rpm for 10 min. Transfer 8 ml of each supernatant with a pipet to plastic tubes. Recentrifuge for 10 min at 3000 rpm. Determine calcium in lOO-fold dilutions by lanthanum chloride diluent of calcium standard solution, and of the supernatant of samples “test” and “reference”. For our atomic absorption spectrophotometer, the settings were as follows: wavelength 211.8 nm; visible range; slit 4; integration time 2 s. The following formula gives the concentration of oxalic acid in mg/l of urine: X 1.05 (Ar -Ax) Ast

1

X 90 X I_11 _ 2oo 40 8

Where Ast, Ar and Ax are absorption measurements of calcium standard, sample “reference” and the supernatant of sample “test” respectively, 90 and 40 are the molecular and atomic weights of oxalic acid and calcium, and 400 the concentration in mg/l of calcium standard; 1.05 is a correction factor based on a constant recovery of 95% of the calcium oxalate; 200 is the constant amount of added oxalate in mg/l. The 24-h excretion of oxalic acid in mg, will be:

1

1181 XAr-Ax-200 Ast Where V is urinary

x v

volume in litres per 24 h.

Results and discussion Collection

of specimen

Most authors [ 5-81 require urinary collection with concentrated HCl in the container, to dissolve calcium oxalate completely and to insure better stability of the sample. Consequently, it is important to acidify not only the sample as Menache describes it [4], but all the 24-h urinary volume. It is imperative that the pH of the urine is less than 1 (if not, acidify with a minimum of concentrated HCl), since it has been proved that at a pH > 1 some oxalic acid remains insoluble [ 31. Technical

variables

1. pH of precipitation

Archer et al. [5] and Hockaday et al. [7] recommend increasing the pH of the sample to 8 to avoid interference by phosphates, but this method is no longer widely used. As we shall demonstrate, this omission does not reduce the percentage of precipitation in the presence of large quantities of phosphates. As in most procedures [5-7,9] pH 5 has been chosen as the optimal pH to precipitate calcium oxalate. 2. Temperature

Boiling the urine as a means of accelerating because it reduces the solubility of magnesium

precipitation is no longer used oxalate [3]. On the other hand,

74 TABLE I RECOVERY

OF OXALIC ACID AS A FUNCTION OF INCREASING AMOUNT OF OXALIC ACID

Amount of oxalic acid solution 4 g/l (in ml)

Overloading in mg of oxalic acid per liter of urine ..~_.__

Radioactivity of reference sample (in cpml

Radioactivity of reference blank (in cpm)

0.1 0.2 0.3 0.4 0.6 0.6

50 100 150 200 250 300 -_

9072 9094 9082 9058 9073 9091

36 34 34 32 36 34

Radioactivity of supernatsnt of sample test fin cpmf --__

Radioactivity of supernatant of blank test (in cpm)

Percentage of oxalic acid recovered (%l

30 32 30 34 30 35

80.1 90.6 92.3 94.9 95.0 94.8

-.-_ 1844 875 719 508 481 489

TABLE II RECOVERY

OF OXALIC ACID AS A FUNCTION OF PRECIPITATION

TIME

Precipitation time (bl

Radioactivity of reference sample (in cpm)

Radioactivity of reference blank fin cpml

Radioactivity of supernatant of sample test (in cpm)

Radioactivity of supernatant of blank test (in cpml

Percentage of oxalic acid recovered (%l

12 24 48 72

8724 8748 8733 8781

32 29 31 32

867 514 454 467

36 34 36 38

90.4 94.5 95.2 95.1

TABLE III EFFECT OF PRINCIPAL INTERFERING Overloading reagents (0.2 ml)

Overloading solution of phosphorus (53.25 g/l) Overloading solution of ~monium nitrogen (58.42 g/D Overloading solution of magnesium (16 g/l) Overloading solution of uric acid (21.32 gll) Reference

SUBSTANCES ON THE OXALIC ACID RECOVERY -~

Amount added (in g/l urine)

Radioaeti~ty of reference sample (in cpm)

Radioactivity of reference blank (m cpml

Radioactivity of supernatant of sample test (in cpml

Ra~oac~~ty of supematant of blank test (in cpml

Percentage of oxalic acid recovered (%l

1.33

8931

29

552

36

94.2

1.46

8963

30

517

34

94.6

0.40

8919

30

558

33

94.1

0.63

8947

34

491

36

94.9

0

8939

31

501

38

94.8

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Hockaday et al. found that boiling causes inaccurately high values [7]. For these reasons, although it takes more time to complete the precipitation, we chose a precipitation temperature of 4” C. 3. Choice of the amount of added calcium Overload of calcium must fullfil two requirements: (a) to allow precipitation of oxalic acid present in the urine and added oxalic acid, even when urinary calcium is at a low concentration, (b) to permit our apparatus to measure urinary calcium such that the amount of absorbance increases linearly with increasing concentration of calcium even at high concentrations. We chose a constant overload of 400 mg of calcium per liter of urine. This allows a determination of 900 mg of total oxalic acid (endogenous + overload) per liter of urine. Under these conditions, linearity is maintained until an endogenous concentration of 600 mg/l is reached. 4. Choice of the amount of added oxalate Koch and Strong [6] showed that the rate of precipitation of calcium oxalate at 4°C is directly proportional to the concentration of oxalate. We studied this rate of precipitation by overloading the 8 ml of urine and 0.4 ml of calcium solution (8 g/l) present in the “reference” and “test” tubes, with increasing quantities of 4 g oxalic acid solution per liter (50 to 300 mg) and with a constant addition of 0.5 ml of [‘4C]oxalic acid solution. The pH of the “test” tube is adjusted to 5.0 and the volumes of both tubes brought to 10 ml. After 48 h at 4°C and the centrifugation step, measurements of the sample “reference” and the supematant of sample “test” radioactivities were performed by mixing 1 ml of these solutions with 10 ml of Unisolve I. The amount of radioactivity for the “blank” (same as the “reference” and “test” tubes, without [ 14C]oxalic acid solution) was also determined. For a given overload in oxalic acid the percentage of precipitated oxalic acid is formulated in the following equation:

[(

Radioactivity of “reference” I( sample [(Radioactivity of

11[(

radioactivity of “reference” blank “reference” sample) -

Radioactivity radioactivity of supernatant of supernatant ( of blank “test” of sample “test” 1 (radioactivity of “reference” blank)]

Results of this investigation are shown in Table I. They confirm ship between precipitation rate and concentration in oxalic acid.

x 100

the relation-

5. Precipitation time Generally, complete precipitation at 4°C is obtained in a time varying from 12 h [ 7,9] to 72 h [6]. We investigated the effect of precipitation time on the percentage of oxalic acid recovered. To this end, we measured the recovery in several urine samples by adding 0.5 ml of [‘4C]oxalic acid solution in addition to all reagents of the standard method, and varying the precipitation time. Measurements of radioactivities, defined in the preceding paragraph, determine the percentage of oxalic acid recovered, for each precipitation time. Results of this study are shown in Table II, and they indicate that after 12 h, 90%

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of the total oxalic acid is recovered. After 24 h, there is 94% recovery, whereas by 48 h or more, it reaches a maximum of 95% precipitation. Consequently, we routinely use a precipitation time of 48 h. However, in an urgent case, the determination can be realised in 24 h, with a slightly inferior percentage+ of recovery.

Accuracy Many substances are known to interfere with the precipitation of calcium oxalate, principally phosphate ions, which may precipitate as calcium phosphate. On the other hand, magnesium ions are known to prevent the complete precipitation of calcium oxalate. We determined the concentration of oxalic acid of a urinary pool overloaded with large quantities of the principal interfering substances. We compared the results of the determinations with those of the non-overloaded pool. For this purpose, we added 0.5 ml of [L4C]oxalic acid solution and 0.2 ml of the different overloading solutions to each urinary sample and to all reagents of the standard method. Table III shows the amount of substances added and the percentage of precipitation in each case. These results demonstrate that the determination of oxalic acid by our method is not modified in the presence of phosphate, ammonium, magnesium or mate ions, even in high concentration. On the other hand, it is more accurate to use a correction factor (1.05) to compensate for the 5% of oxalic acid remaining in the solution because in all urine samples tested, 95% of the oxalic acid was precipitated regardless of the initial concentration of oxalic acid.

The study of the repeatability for two urine samples in low (A) and high (B) concentrations gave the following results: Urine A: average of 30 determinations = 17 mg/l, S.D. = 2.95 mg/l. Urine B: average of 30 determinations = ,186 mg/l, S.D. = 6.10 mg/l. On the other hand, for our laboratory quality control, we set up a pool of acidified urines, divided into fractions of 20 ml and frozen at -20°C. The concentration of oxalic acid in this pool was measured twice a week for 15 weeks. The average value was determined to be 26 mg/l, with a standard deviation of 3.45 mg/l. Normal values 30 healthy adults of both sexes, from 19 diet, were found to have an average excretion deviation of 9 mg. Consequently, daily urinary studied is between 2 and 38 mg (average + those given by most authors [2-4,6,7],

to 40 years old, on a “normal” of 20 mg/24 h, with a standard output of oxalic acid of subjects 2 S.D.). These norms agree with

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Conclusion Our method for determining oxalic acid is simpler than most existing procedures because it measures calcium remaining in solution, and not calcium precipitated in the form of oxalate. The detailed study of various technical parameters allows a specific and constant precipitation of oxalic acid in urine. Therefore this assay provides a useful and reliable method for the determination of oxalic acid in urine. References 1 Hodgkinson. A. (1970) (Review) CUn. Chem. 16.647-567 2 Fraser, J. and Campbell, D.J. (1972) Clin. Biochem. 6.00-104 3 Henry, R.J. (1974) Clintcal Chemistry, Principles end Technics (Henry. R.J., Cannon, D.C., Winkelman. J.W.. eds.). p. 1361, Harper and Row, New York 4 Menache, R. (1974) Clin. Chem. 20.1444-1446 6 Archer, H.. Dormer-Scowen. E. and Watts, R. (1967) Clin. Sci. 16. 405-411 6 Koch, G.H. and Strong, F.M. (1060) Anal. Biochem. 27.162-171 7 Hockaday. D.R.. Frederick, E.W.. Clayton, J.E. and Smith, L.H. (1966) J. Lab. Clin. Med. 65.677687 S Giterson. A.L.. Slooff, P.A.M. and Schouten, H. (1970) Clin. Chim. Acta 29. 342-343 0 Mayer. G.G., Markow, D. and Karp, F. (1963) Clin. Chem. 9.334-339