ANALYTICAL

BIOCHEMISTRY

94,470-476

(1979)

An Electron-Capture Gas Chromatographic Procedure Estimation of Oxalic Acid in Urine D.J.Tocc0,A.E.W. Merck

Institute

DUNCAN, R.M.

for Therapeutic Research, Merck & Company, Inc.,

NOLL,AND

Merck Sharp & Dohme West Point, Pennsylvania

D.E. Research 19486

for the

DUGGAN Laboratories,

Received November 20, 1978 An analytical procedure for the estimation of urinary oxalate is described which satisfies the requirements of specificity, recovery, and negligibIe generation of oxalate from progenitors. The procedure involves precipitation of oxalate from urine as the calcium salt and subsequent diesterification with 2chloroethanol. The derivative is detected by electron capture-gas chromatography with [Wloxalate used as a recovery standard. The electron-capture response is linear over the range of 5 to 40 pg carried through the procedure. The coefficient of variation in replicate aliquots over the entire range is 7%. Total urinary oxalate excretion for the periods 0800-1200, 1200-1600, and 1600-0800 h on the following day were determined for each of eight volunteers over periods of 5 or 7 days. The mean excretion of oxalate was 35.6 2 11.9 mg/day. There was no suggestion of a diurnal pattern in oxalate excretion. Oxalate levels in dog plasma, as determined by the electron capture-gas chromatographic procedure, were high (-0.7 &ml) compared to theoretical oxalate levels (0.1 to 0.2 &ml) as estimated from the renal clearance of [lsC]oxalate or [Yloxalate. These data clearly indicate that oxalogenesis occurs during the assay of plasma, even under the mildest of separation conditions.

A number of analytical procedures for the estimation of urinary oxalate have been developed and several review articles on these methods have been published (l-3). In general, the methods can be divided into four groups: direct precipitation, solvent extraction, isotope dilution, and enzymatic procedures (1). Most methods for oxalate are subject to three sources of error: lack of quantitative recovery, lack of specificity, and artifactual generation of oxalate during the analytical procedure. Recovery has frequently been corrected by employing [‘“Cl oxalate as an internal standard and specificity has been ensured using enzymatic methods (2,5) or chromatography (4). The problem of oxalogenesis, conversion of carbohydrates and other substances to oxalic acid, has not been dealt with satisfactorily and in most methods for oxalate determination, not even considered. Thus the wide range of values for urinary oxalate reported in the 0003-2697/79/060470-07$02.00/O Copyright All rights

63 1979 by Academic Press, Inc. of reproduction in any form reserved.

470

literature and summarized by Hallson and Rose (2) and still wider range of values for plasma oxalate (1) suggest that oxalogenesis could be operative when the values are high (40-50 mg/day) and poor recovery could be suspect when the values are low (3-5 mg/day). Described is an electron capture-gas chromatographic procedure for oxalate determinations in urine which satisfies the requirements of specificity, good recovery, and negligible oxalogenesis. The accuracy and specificity of the method has been verified by a second procedure employing the technique of GC-MS with selective ion monitoring (6). METHODS

The analytical procedure for the estimation of urinary oxalic acid involves copre1 Abbreviations used: GC-MS, Gas chromatography-mass spectometry; EC-GC, electron capturegas chromatography.

OXALIC

ACID

BY ELECTRON

cipitation of oxalic acid and added [14C]oxalate, which serves as an internal standard. A chlorinated derivative of oxalic acid is prepared which is extracted and detected by electron capture-gas chromatography. Recovery of oxalate, in this one-tube assay, is determined by radiometry. Reagents. Saturated CaSO,*2H,O (ca. 2.5 mg/ml water). [14C]Oxalate recovery standard (AmershamSearle): 1 PCi (74 mCi/mmol) dissolved in 2.0 ml 0.01 N HCl, prepared fresh. Boron trichloride-2-chloroethanol (lo%, w/v) (Applied Sciences). Lindane injection standard: 0.4 pg lindane/ ml cyclohexane- 1% ethanol. GC conditions. A Hewlett-Packard 5840 A gas chromatograph equipped with automated sampler and electron-capture detector was used. The detector and injection port temperatures were 300 and 25O”C, respectively. Oven temperature was programmed at 150°C for 12 min and then increased 25”C/min to 250°C for 2 min. A 6-ft x ?&in.-i.d. glass column was packed with 1% OV-17 on Gas-Chrom Q. Carrier gas was argon-methane, 90/10 with a flow rate of 150 ml/min. The microprocessor unit of the instrument was used to control chromatographic conditions and determine peak areas. PROCEDURE

To 1 .O ml urine in a 13-ml glass-stoppered centrifuge tube is added 0.1 ml [14C]oxalate recovery standard. After mixing, 2.0 ml absolute ethanol and 0.5 ml CaSO, . 2Hz0 reagent are added, mixed, and placed in an ice bath for 18 h. The samples are centrifuged for 20 min and the supernatant is drained. The pellet is washed with 1.0 ml absolute ethanol and centrifuged 20 min. The supematant is drained and the pellet dried under oil pump vacuum (1 mm Hg) for about 30 min at room temperature. To the dried pellet is added 0.5 ml of borontrichloride-2-chloroethanol reagent and the sample stoppered tightly and heated in a

471

CAPTURE

100°C dry-block for 1 h. After cooling to room temperature, 10 ml of cyclohexanelindane reagent is added and shaken mechanically for 10 min. One milliliter of water is added and shaken for 1 additional min. The samples are centrifuged and the organic phase is transferred to a clean tube. In the absence of water, the derivative is stable for at least 2 days. Radioactivity in a l.Oml aliquot of the organic phase and a O.lml aliquot of the [14C]oxalate injection standard is determined by liquid scintillation counting. Another aliquot (5 ~1) is injected into the gas chromatograph. The ratio oxalate peak area/lindane peak area is corrected for the recovery of [14C]oxalate. Correction for the contribution of labeled oxalate to total area can be omitted from most urine samples since it represents approximately 1% of urine oxalate levels. Water standards of 5, 10, 20, and 40 pg of oxalate are carried through the procedure and urine oxalate levels are read off the water standard curve. Correlation of electron-capture procedure with GC-MS procedure. The electron-

capture procedure described in this report and the GC-MS procedure (6) were applied to urine samples from eight normal subjects. Urine samples were collected and aliquots immediately assayed by the two methods. Oxalate

excretion

in normal

subjects.

During the pretreatment phase of a controlled clinical study, urine was collected daily from eight subjects over periods of 5 or 7 days. The time periods of collection were 0800-1200, 1200-1600, and 16000800 h. During the collection period, subjects were instructed to avoid alcohol and foods rich in oxalate. The urine collections were precisely timed, volumes noted, and the specimens immediately frozen. After thawing by immersion in water at room temperature, to each specimen tube containing 10 ml urine was added 0.5 ml of 2~ HCl. The tubes were restoppered, shaken mechanically for 10 min, then ad-

472

TOCCO ET AL.

FIG. 1. Electron capture-gas chromatogam of a human urine sample. Oxalic acid (15 &ml) at 2.8 min and the injection standard, lindane, at 9.31 min.

peaked

OXALIC

FIG. 2. Oxalate

IO 20 OXALIC ACID, dichloroethyl

ester

ACID

BY ELECTRON

40 erg standard

curve.

justed with base to the original pH. An aliquot equivalent to 1.0 ml of the original urine sample was assayed. RESULTS

AND DISCUSSION

Figure 1 shows a typical chromatogram obtained when human urine was processed through the analytical procedure. Oxalate as the acyl derivative had a retention time of 2.80 min and lindane injection standard, 9.31 min. The esterification of oxalate was examined between 30 and 60 min in a boiling water bath. In all samples, the same relative peak area was obtained suggesting that the reaction remained constant within this time period. Earlier time periods, 10 and 20 min, gave an incomplete reaction as evidenced by a lower oxalate peak. The calibration curve for the dichloroethyl derivative of oxalate obtained by plotting the ratio of the peak area of the oxalate peak to that of the injection standard, corrected for [14C]oxalate recovery, is shown in Fig. 2. The curve is linear over the range of S-40 Fg. The coefficient of variation is 7% for five replicate aliquots at each point over the range of 5 to 40 pg. Urine samples

473

CAPTURE

from eight normal subjects were assayed by the EC-GC and GC-MS (6) procedures. The correlation between the two methods was excellent (r = .9918, n = 8) as shown in Fig. 3. Several reports indicate the production of oxalate from natural substances in urine (7). We have recognized the significance of oxalogenesis when we attempted to apply the EC-GC and GC-MS methods for urinary oxalate to plasma samples. Aliquots of heparinized dog plasma were spiked with [ 13C]oxalate or [ 14C]oxalate and filtered through Amicon ultrafilter cones. Application of the GC-MS and the ECGC procedures without further treatment gave values which averaged 0.73 and 0.65 pg/ml, respectively (Table 1). After treatment with heat (100°C water bath for 10 min) or passing the sample through Dowex-3 Cl- columns and eluting with HCl, the values were increased to 1.34 and 24.6 by the GCMS method and 1.10 and 55.2 by the EC-GC method, respectively. The variability observed in the four samples assayed by the two methods following anion-exchange treatment suggests that one should be cautious in employing isolation techniques in the development of methodology for oxalate in plasma. 60 -

50 -

T 40. EC 30: ,”

20-

‘OliLL-IO

FIG. 3. Correlation ses of human samples

20

30 GC-MS

of EC-GC (n = 8).

40 (pg/ml) with

50

GC-MS

60 analy-

474

TOCCO

ET AL.

Immediately following withdrawal of plasma from the dog in the above experiment, renal and plasma clearances were determined. Following an iv bolus of [14C]oxalate, plasma and urine were sampled sequentially through 5 h. The mean of six incremental renal clearances was 51.8 ml/ min, in excellent agreement with plasma clearance of 53.0 mUmin calculated from V,*K,,3 (Fig. 4). Mean urinary excretion of oxalate was 7.2 pg*min-‘. Plasma oxalate levels estimated from the equation:

TABLE

EFFECTOFVARIOUSTREATMENTS ON ULTRAFILTEREDPLASMA OXALATE VALUES" GC-MS Sample number

Treatment

C,=uV, V ClJ

EC-GC

pg/rnl

Mean

@ml

None

1 2

0.74 0.72

0.73

0.48 0.82

0.65

Heat

1 2

1.32 1.35

1.34

1.25 0.95

1.10

Anion exchange

1 2

21.8 27.3

24.6

39.7 70.8

I 100

1

.

200

300

MINUTES

FIG. 4. A 22.5-kg Plasma

was sampled

female beagle received as indicated, and urine

Mean

55.2

n Aliquots of heparinized dog plasma were spiked with [Wloxalate (5 &ml) or [Wloxalate (40 &ml) and filtered through Amicon ultrafilter cones. After treatment with heat (lOO°C water bath/l0 min) or adsorption onto a Dowex 3 Cl- column followed by washing and elution with 0.5 M HCI, the samples were assayed for oxalate by GC-MS or EC-W methods.

where C, = plasma oxalate, (pg/ml), UV = urinary oxalate, (pg. min-‘, and Vcl,r = renal clearance, (ml *min-‘) , indicated that the concentration of oxalate in the plasma was 0.15 pg/ml. Estimated plasma

100

1

a bolus collected

injection of 5.8 pg [‘4C]oxalate (25 &ii~mol). at 30-min intervals through 5 h.

OXALIC

ACID BY ELECTRON TABLE

CONCENTRATION

OF OXALATE

IN URINE

475

CAPTURE

2

BEFORE AND AFTER OXALATE

DECARBOXYLASE

TREATMENT

Urine sample Treatment

1

2

3

4

None (fresh urine) 5-hr incubation (25°C) Oxalate decarboxylase (% decarboxylated) Theoretical” Excess “Oxalogenesis” (%)

16.95 15.75 1.49 (99.91) 0.22 1.27 (7.5)

12.8 14.2 2.8 (99.73) 3.5

22.2 22.2 4.3 (99.81) 0.9 3.4 (15.3)

25.5 25.5 9.3 (99.W 2.3 2.3 (9.0)

(i

a Theoretical residual oxalate = oxalate in fresh urine (100 - percentage decarboxylated).

oxalate values based upon renal clearances in five other experiments (6) was 0.126 + 0.049 pg*rnl-’ (range 0.048-0.177). This value, approximately one-fourth the lowest value obtained by the EC-GC method, suggests that during the EC-GC analytical procedure oxalogenesis had occurred. Thus, even under the mildest experimental conditions employed, significant artifactual generation of oxalate from progenitors endogenous to plasma occurs, and where ionexchange is employed [e.g. Ref. (8)], massive oxalogenesis occurs. Oxalogenesis during the EC-GC assay of human urine was minimal ranging from 0 to 15.3% (Table 2). Urine samples from TABLE

3

EXCRETION OF OXALATE IN URINE OF NORMAL SUBJECTS DURING A 5- OR ~-DAY PERIOD

Subject 1 2 3 4 5 6 7 8 Grand mean

Days in study 7 5 5 7 7 5 7 5

Oxalate excreted (mg/day 2 SD) 27.1 37.9 39.4 51.8 15.1 48.5 28.3 36.9 35.6

f 6.5 f 24.2 zk 10.3 2 7.7 + 4.1 k 17.5 k 6.9 2 14.2 ” 11.9

Coefficient of variation m 24 64 26 15 27 36 24 38 34

four subjects were incubated with 1 unit of oxalate decarboxylase (Sigma Chemical Co.) for 5 h. Using [14C]oxalate as an internal standard, it was determined that 99.73 to 99.91% of the urinary oxalate had been decarboxylated. Subsequent assay yielded an apparent oxalate value of 0.9 to 3.5 j&ml or oxalogenesis equivalent to 15.3% or less of the original oxalate concentration. Aqueous solutions of the following substances exhibited no interference in the analysis: citric, 100 ,ug/ml; oxaloacetic, 50 pg/ml; succinic, 50 pg/ml; pyruvic, 20 pg/ ml; glycolic, 20 &ml; and glyoxylic, 20 &ml. Urinary oxalate excretion: The mean values of daily urinary oxalate excretion ranged from 15.1 (Subject 5) to 51.8 mg/day (Subject 4) for 8 normal subjects over a 5 to 7-‘day period (Table 3); i.e., an interindividual coefficient of variation of 34%. Intraindividual variation ranged from 15.1% (Subject 4) to 64% (Subject 2). In Fig. 5 are plotted urinary excretion rates for four of these subjects as a function of time of day. The collection periods were 0800-1200, 1200-1600, and 16000800 h the following day. It is apparent from these data that there is no suggestion of a diurnal pattern in oxalate excretion. The distribution of oxalate excretion rates by all subjects is depicted in Figure 6. Most specimens (79%) indicated an excretion

476

TOCCO ET AL. SUBJECT

7

SUBJECT

5

SUBJECT

4

SUBJECT

I

2

DAY

FIG. 5. Urinary excretion rates for oxalate as functions of time of day.

rate of 0.5 to 2.0 mg/h. Approximately 15% of the specimens indicated excretion rates greater than this range and 6% less than this range. REFERENCES 1. Hodgkinson, A. (1970) C/in. Chem. 16, 547. 2. Hallson, P., and Rose, G. A. (1974) Clin. Chem. Acta

55, 29.

3. Ito, H. (1975) Japan J. Ural. 66, 19. 4. Charransol, Cl., and Desgraz, P. (1970) J. Chromatogr.

1

I:0

I15

h

I I * 2.0 2.5 3.0

3.5

4.0

mg PER HR

FIG. 6. Distribution rates (n = 144).

of observed oxalate excretion

48, 530.

5. Ribeiro, M., and Elliot, J. S. (1964) Invest. Ural. 2, 78. 6. Duggan, D. E., Walker, R. W., Noll, R. M., and VandenHeuvel, W. J. A. (1979)Anal. Biochem. 94, 477-482. 7. Hodgkinson, A., and Zarembski, P. M. (1961) Ahalyst

86, 16.

8. Krugers Dagneaux, P. G. L. C., Klein Elhorst, J. T., and Olthuis, R. M. F. G. (1976) Clin. Chem. Acta 71, 319.