Clin. Biochem. 5, 99-103 (1972)

INDIRECT MEASURE OF OXALIC ACID IN U R I N E BY ATOMIC ABSORPTION SPECTROPHOTOMETRY

J. FRASER and D. J. CAMPBELL*

Department of Clinical Pathology, University Hospital, Edmonton, Alberta

(Received August 1, 1971)

SUMMARY

1. Oxalic acid is precipitated f rom urine along with a known addition of sodium oxalate, by an excess of calcium chloride at a pH of 5, and a t empera ture of 4 degrees. The precipi tated calcium oxalate solution is then dissolved in H~S04.

2. The calcium content is then determined by comparison with s tandards by atomic absorption, and by calculation the results are expressed as mil- l igrams oxalic acid dihydrate per 24 h urine volume.

A number of methods for the determinat ion of oxalic acid have evolved over the years to include 4 groups: (a) direct precipitation, (b) solvent extraction, (c) isotope dilution, and (d) enzymatic, as well as various combinations of these methods. The method of Zaremski and Hodgkinson (1) appears to give excellent results but the procedure was considered too involved for a screening study.

Only the direct precipitat ion method has been employed in this hospital. Koch and Strong (2) proved the necessity of the addition of a known quant i ty of oxalate, in a precipitat ion method, as well as the desirabil i ty of washing the oxalate precipi tate with a wa te r sa tura ted with calcium oxalate.

Since the completion of this work, Giterson et al (3) have published a method which has the similari ty of determining the calcium but uses a smaller sample and is less t ime consuming since there is no washing or re-dissolving of precipi tate as the superna tan t is measured for excess calcium.

*Correspondence: Dr. D. J. Campbell, Head Division of Clinical Chemistry, Department of Pathology, Vancouver General Hospital, Vancouver 9, B.C.

100 F R A S E R & C A M P B E L L

The method of Daniels et al (4) had been in use for some time for determination of urine oxalic but when a s tudy was to be undertaken in- volving a great number of specimens, elimination of the t i t ra t ion step of the procedure was sought because of the possible errors involved in oxida- tion-reduction reactions of urine, and the inconvenience of multiple t i t ra- tions over s teaming hot water. The proposed method was therefore developed to determine the calcium of the calcium oxalate precipitate using atomic absorption.

MATERIALS AND METHODS:

Apparatus: Special 100 ml pear-shaped centrifuge tubes (Pyrex 8200) with 10 mm o.d. x 40 mm stem were used for precipitation and washing of calcium oxalate. Internat ional centrifuge with head No. 246 and shield No. 395. Tall-form 250 ml beaker (Pyrex 1140) served as a convenient support for the tubes. Atomic absorption spectrophotometer, Perkin-Elmer Model 303 equipped with a hollow cathode single element Calcium lamp.

Reagents: Sodium 0xalate, 2 mg/ml Sodium oxalate, 0.2 g deionized water q.s., 100 ml Calcium Chloride 10~ (w/v) Ammonium hydroxide Calcium oxalate saturated wash water.

1 q powdered calcium oxalate in 2000 ml H_~O add solution of 2 mg pentachlorophenal in 1 ml ethanol Mix for 24 hours, allow to settle, and f i l ter through a sintered glass fi l ter funnel containing an asbestos pad.

Calcium stock standard, 1 mg/ml

Calcium Final conc. ml of volume

mg/lO0 stock with ml standard water

2.5 5 200 5 5 100 7.5 15 200 10 10 i00

12.5 25 200 15 15 100

URINE OXALATE 101

PROCEDURE

S a m p l e P r e p a r a t i o n :

Collect a 24 h urine in a bottle containing 10 ml of concentrated HC1 and refr igerate until ready to analyze. Centr i fuge 250 ml of the acidified urine and then adjust pH to 5 by addition of 6 M ammonia, using a pH meter and magnetic st irrer. Duplicate 100 ml portions of urine are measured into pear shaped centr ifuge tubes (Pyrex 8200 supported in tall form 250 ml beaker Pyrex 1140) and exactly 2 ml. of 2 mg /ml sodium oxalate added to each and contents mixed. If the urine oxalic acid is known to exceed 50 mg/ l i t re , the NmC~04 addition is omitted. Then 2 ml of 10'S CaCh • 2H20 is added and the tube covered and mixed. The samples are refr igerated at 4 ° for 2 full days (64 hours more convenient) and then centrifuged at 2000 rpm for 15 min. The supernatant is removed by suc- tion (it may be necessary to remove about half the urine and spin a second time before removing the last of the urine). Wash the wall of the centri- fuge tube with about 5 ml wash water, resuspend the precipitate using a s t i r r ing rod, wash the rod, swirl the tube and again wash down the sides. Centr i fuge for 10 minutes, remove supernatant with suction and repeat wash procedure once more (may be left in the ref r igera tor at this point, if necessary).

Add 2 ml of 1 N H~SO, to each tube, mix on Vortex, and heat in 80°C water bath to dissolve precipitate. Transfer quanti tat ively to 25 ml volume- tric flask and dilute to volume with water. C a l c i u m D e t e r m i n a t i o n - - Dilute 1 ml of s tandards and samples to 25 ml with water (add 1 mil of a 2 ml in 25 ml dilution of 1 N H~SO,) to each of the s tandards so as to contain acid equal to tha t in the samples. I n s t r u m e n t a l S e t t i n g s - -

Boiling burner Wavelength - - sett ing at 211.8 ram. Range - - Vis. (423 nm) Slit 4 Lamp current 10 ma Scale 2 Air 30 psi - - 9 flow Acetylene 8 psi - - 11.5 flow (yellow flame) Sample flow 4 - 5 ml /min

Calcu la t i ons :

On one-cycle semilog graph paper plot ~, absorption against s tandard concentration. Read Ca conc. of samples f rom the graph and apply the factor below to correct for concentration of urine f rom 100 ml to 25 ml, to convert calcium to oxalic acid dihydrate, subtract the known addition, and convert to per 24 hour volume.

102 FRASER & CAMPBELL

[ ( 25 126 lO00~ x x y ~ x 7-~ x ioo/

V [(X X 7.875) -- S] X 1000

V - S X ~ which equals

X = mg Ca/100 ml (with or without added Na~C,0,). S = mg oxalic acid dihydrate added as Na,C,04

(38 mg/ l if 4 mg Na2C,O, added to 100 ml of urine). V = volume of 24 hour urine in ml

RESULTS

In preparing the s tandards the diluent typically in use for calcium in biological specimens, namely lan thanum (or s t ront ium) has purposely been left out as it was shown to inhibit the response f rom standards which included oxalate along with the calcium.

Calculations have been based on the theoretical quant i ty of oxalic which should be present for the calcium determined, which appears substant iated by the urine recovery series. This was necessitated by the consistently low recovery of an aqueous s tandard. A 100 ml portion of a 50 mg/1 oxalic acid dihydrate aqueous s tandard gave recoveries of only 75 - 9 0 7 .

A brief comparison of samples determined by three method variat ions yielded three levels of results with the presented method in the middle (Table 1).

Possible explanations of the differences in results with each method could include :

Method (b) low due to incomplete precipitation of the urine oxalate and a loss in the precipitate because of the ammonia wash.

Method (c) high due to oxidation-reduction reactions of urine compo- nents other than oxalate. A series of recoveries using the presented method gave percentages of 80 - 115 (Table 2).

Reproducibility of the method was evaluated by assaying a pooled urine. One s tandard deviation was ___ 2.1 for a mean value of 24.5 mg/1 calculat- ed f rom 29 duplicate determinations.

A very limited number of "normal" urines collected f rom hospitalized patients with no his tory of kidney stones yielded a normal range of 13 - 36 m g / d a y on seven specimens with a mean value of 20 milligrams. These values are slightly higher than Hodgkinson (5) who quotes daily excretion of oxalic acid (anhydrous) ranges between 10 and 40 mg with a mean value of about 20 milligrams.

U R I N E OXALATE 103

TABLE 1

COMPARISON OF RESULTS BY THREE METHODS

Method (a) Method (b) Method (c)

16 6 17 5 36 14 16 9

16 < 5 23 7 29 12

13 < 5 5 < 5

< 5 < 5

23 11 41 18 29 12

TABLE 2

OXALIC ACID DIHYDRATE RECOVERY FROM URINE

Urine Concentration Added Found Recovery

mg/1 mg/l mg/l %

5 7 12 100 7 7 13 86

24 25 49 100 0 12.5 10 80 0 25 20 80 0 32.5 33 102 3 25 27 b6 3 32.5 33 92

18 7 22 88 0.6 32.5 38 115

62 38 101 103

20 21, 35 24 32 37 33

15 29 20 48 21 37 25 62

Method Method Method

(a) is as presented (b) Daniels method (4) (c) Koch method (2) except ppl.

dissolved in sulphuric acid and titrated with potassium per- manganate.

The lower results of method (b) appear fu r the r substant iated in tha t a normal range calculated f rom eleven specimens is 5 - 18 m g / d a y with a mean value of 11 milligrams.

DISCUSSION

With the availability of an atomic absorption spectrophotometer we have been able to eliminate t i t ra t ion and because of complete precipitation, the results have been more reproducible for the determinat ion of oxalic acid dihydrate in urine than previous procedures.

REFERENCES 1. ZAREMBSKI, P. M. and HODGKINSON, A. The Fluorimetric determination of

oxalic acid in blood and other biological materials. Biochem. J. 96, 717-721 (1965}. ~. KOCH, G. H. and STRONG, F. M, Determination of Oxalate in Urine, Anal. Biochem.

27, 162-171, (1969). $. GITERSON, A. L., STOOFF, P. A. M. and SCHAUTER, H. Oxalate in Urine, Clin.

Chim. Acta, 29, 342-343, (1970}. $. DANIELS, R. A., MICHELS, R., AISEN, P. and GOLDSTEIN, G. Familial Hyper-

oxaluria, Am. J. Med., 29, 820-831, (1960). 5. HODGKINSON, A. Determination of Oxalic Acid in Biological Material, Clin. Chim.

16, 547-555, (1970}.