Serum oxalate in human beings and rats as determined with the use of ion chromatography

Serum oxalate in human beings and rats as determined with the use of ion chromatography ANASTASIA H. HARRIS, ROBERT W. FREEL, and MARGUERITE HATCH GAINESVILLE, FLORIDA

Previous enzymatic determinations have suggested that serum oxalate concentrations in normal rats, the main animal model used in urolithiasis research, to be 3 to 5 times greater than those in healthy human subjects. In this report we validated this observation using a different method (ion chromatography) on serum samples from healthy rats and human subjects that were prepared and handled similarly. Oxalate recoveries during sample preparation for ion chromatography were strongly and variably affected by ultrafiltration devices employed for sample deproteinization and after Clⴚ removal by means of ion exchange. When oxalate recoveries were accounted for, we found significant differences in serum oxalate (6 human samples, 1.47 ⴞ 0.15 ␮mol/L; and 15 rat samples, 9.88 ⴞ 0.91 ␮mol/L). We conclude that ion-chromatographic techniques confirm the differences in serum oxalate concentrations between rats and human beings measured enzymatically and that failure to account for oxalate losses during sample preparation for ion chromatography can lead to significant underestimation of serum oxalate in both species. (J Lab Clin Med 2004;144:45-52)

T

he determination of serum or plasma oxalate concentrations in mammals is of special significance in studies of oxalate homeostasis because this dicarboxylic anion is an important component in the formation of calcium oxalate stones in renal tissue.1– 4 Levels of serum oxalate in healthy human subjects are now routinely reported to be in the range of 0.3 to 5 ␮mol/L rather than the submillimolar range,1,3,5–10 as was suggested by the findings of earlier studies. This evolution in accepted concentrations of serum oxalate in human beings is a consequence of refined analytical methods,1,3,8 –11 as well as an increased appreciation of the importance of sample handling and preparation From the Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine. Supported by National Institutes of Health grants R01 DK56245-02 and 2 R42 DK55944-02. Submitted for publication February 5, 2004; revision submitted April 16, 2004; accepted April 27, 2004. Reprint requests: Marguerite Hatch, PhD, Department of Pathology, Immunology, and Laboratory Medicine, P.O. Box 100275, 1600 SW Archer Road, Gainesville, FL 32610; e-mail: [email protected]. © 2004 Elsevier Inc. All rights reserved. 0022-2143/$ – see front matter doi:10.1016/j.lab.2004.04.008

before analysis.1,10,11 These refinements in serum oxalate determination have led, in our estimate, to the notion that lower levels are “more correct.” Although this may be true, it would seem imperative that oxalate generation or loss be monitored throughout the samplepreparation procedure for any analytical method. In contrast to the many reports of oxalate concentrations in human serum, relatively few studies have been conducted to measure serum oxalate in nonhuman mammals—a significant shortcoming, given that many experimental studies on renal stone formation rely on rodent models of oxalate disease states. When oxalate levels in rat serum have been reported2,12–14 these values have tended to be several times greater than accepted levels in healthy human subjects.1,3,6 –10 Given that rat serum oxalate concentrations have not been extensively reported, it is possible that accepted serum oxalate levels in these animals will decrease with greater scrutiny, which happened with regard to human serum. Small decreases in rat serum oxalate concentrations are trivial in an absolute or osmotic sense but can significantly affect conclusions regarding the renal handling of oxalate obtained from renal-clearance studies.12,14 In this report we made a comparison of serum oxalate 45

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concentrations in healthy human subjects and rats using similar sample-preparation and analytical (ion-chromatography) methods, plus routine monitoring of oxalate losses throughout the sample-handling process. We conclude that oxalate recovery is an important factor to be considered in the preparation of samples for analysis for either species. Failure to account for losses can lead to significant underestimation of serum oxalate concentrations. Furthermore, this direct comparison involving an ion-chromatographic technique indicates clearly that oxalate concentrations in rat serum are indeed several times greater than those in human beings. METHODS Ion chromatography. Ion chromatography was performed with the use of a DX120 chromatography system with an IonPac AS11 analytical column (4 ⫻ 250 mm) and an IonPac AG11 guard column (4 ⫻ 50 mm) (Dionex, Sunnyvale, Calif). Eluent strength and flow rate were optimized separately for oxalate peak resolution in human- and ratserum matrixes. For human serum, the eluent was 24 mmol/L sodium tetraborate and 24 mmol/L boric acid (pH 9.3), with a flow rate of 0.80 ml/min; for rat serum it was 16.5 mmol/L sodium tetraborate and 16.5 mmol/L boric acid (pH 9.3), with a flow rate of 1.0 ml/min. We suppressed background conductivity with the use of 15 mmol/L sulfuric acid pumped at a rate of 8 mL/min through an ASRS-Ultra (anion selfregenerating suppressor) without current (micromembrane suppressor mode). Detection was carried out with the use of conductivity at 35°C. We used a 100-␮L injection loop; it was flushed with 2 mL of purified water between injections. Data were acquired over 25 to 35 minutes per sample injection and processed with the use of PeakNet 6.0 software (Dionex). We prepared standards using oxalic acid dihydrate. The lower limit of reliable detection was 0.25 ␮mol/L oxalate. Serum preparation for ion chromatography. Blood samples were obtained, without control of dietary intake, from 6 nonfasting, apparently healthy adults (3 male, 3 female). Informed consent was obtained from these volunteers under a protocol approved by the University of Florida Institutional Review Board, and the research was carried out in accordance with the principles of the Declaration of Helsinki. Blood was obtained from 15 nonfasting male Sprague-Dawley rats (546865 g) given free access to food (Teklad Diet-8604; Harlan, Indianapolis, Ind) and water. Each rat was killed with an overdose of anesthetic (150 mg sodium pentobarbital/kg body wt) before blood was drawn by means of cardiac puncture. Blood was drawn into prechilled BD Vacutainer SST Gel and Clot Activator tubes (Becton Dickinson, Franklin Lakes, NJ), which were immediately placed on ice. Serum was obtained by means of centrifugation at 3000g and 4°C for 20 minutes. We dedicated 1 mL of each serum sample to the monitoring of oxalate losses incurred throughout the sample preparation by adding carbon 14 –labeled oxalic acid (0.4 ␮Ci/mL; New England Nuclear, Boston, Mass) and processing it in parallel with the remainder of the serum for ionchromatography measurements. At each step of sample pro-

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cessing, oxalate losses were determined with the use of liquid-scintillation spectrometry. We ultrafiltered all serum samples in 1-mL aliquots using Amicon Ultra-4 devices at 3200g and 4°C for 30 minutes and acidified them by collecting them in 50 mg of washed hydrogen form resin (see below) placed in the ultrafiltrate-collection receptacle before centrifugation. Before use, the Ultra-4 devices were rinsed with 0.1N NaOH and then water, followed by centrifugation with water, in accordance with the manufacturer’s instructions. Acidified serum ultrafiltrate was assayed immediately or stored at ⫺80°C for a maximum of 1 week. In a series of determinations intended to evaluate the impact of chloride interference on ion-chromatographic detection of oxalate, we removed chloride from ultrafiltrate immediately before chromatography by adding 150 mg of silver-form resin to 750 ␮L of the acidified ultrafiltrate. The acidified ultrafiltrate sample was vigorously vortexed with resin for 30 seconds and centrifuged at 14,000g for 15 seconds as a means of pelleting the resin. A 500-␮L sample was then applied to the injection valve with 100 ␮L of this sample being injected onto the column. Preparation of ion-exchange resins. We prepared the hydrogen form of the cation-exchange resin AG 50W-X8 (Bio-Rad Laboratories, Hercules, Calif) before use by mixing 10 g of resin in 500 mL of 1N HCl for 30 minutes. We made the silver form of the cation-exchange resin by mixing the hydrogen-form cation-exchange resin, 10 g, in 200 mL of 0.1 mol/L silver nitrate for 30 minutes. Both forms of resin were rinsed thoroughly with water, air-dried, and stored in airtight polystyrene containers in the dark. All of the chemicals used were of reagent grade and were obtained from Sigma-Aldrich (St Louis, MO). The water used to make solutions was freshly drawn from a Barnstead Nanopure water system (resistivity ⬎ 18 megaohms-cm) (Barnstead International, Dubuque, Iowa). Comparison of ultrafiltration devices. One experimental series involved a comparison of devices commonly used in the preparation of serum ultrafiltrates for subsequent oxalate analysis by means of various methods, including ion chromatography. The following devices were used: Amicon Centriflo CF25 (Amicon, Inc, Beverly, Mass)2,5,8 –14; Millipore Amicon Ultra-4, Millipore Centrifree,15–17 Millipore Microcon10, Millipore Microcon-30, and Millipore Ultrafree-MC with Ultracell-PL membrane (Millipore Corp, Bedford, Mass); Pall Nanosep (Pall Life Sciences, Ann Arbor, Mich); and Sartorius Centrisart I (Sartorius, Goettingen, Germany).4,7,18 Blood samples were collected from 6 apparently healthy adults and treated as described above for the collection of serum. Each serum sample was spiked with carbon 14 – labeled oxalic acid (0.4 ␮Ci/mL) as tracer to reveal losses of oxalate and aliquoted into the ultrafiltration devices as follows: 1 mL into the Apollo, Centriflo CF25, Centrisart, and Ultra-4 devices; 500 ␮L into the Centrifree devices; 200 ␮L into the Microcon and Nanosep devices; and 120 ␮L into the Ultrafree-MC devices. We prepared an additional aliquot in accordance with a method reported by Marangella19 by adding 40 ␮L of concentrated hydrochloric acid to 1 mL of serum, then vortexing the mixture for 6 minutes before ultrafiltering 500 ␮L of the serum mixture with the Centrifree

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device. Centrifugation was carried out at 4°C for 30 minutes. The relative centrifugal force applied to each device was as recommended by the manufacturer: 14,000g for Microcon and Nanosep, 7,500g for Apollo and Ultra-4, 5000g for Ultrafree-MC, 2,000g for Centriflo CF25 and Centrifree, and 1,250g during first 5 minutes, followed by 2,500g during the next 25 minutes, for the Centrisart I. Each serum sample was processed in duplicate through each device. Statistical methods. Results are presented as mean ⫾ SEM for the number of samples. We carried out multiple comparisons using 1-way analysis of variance with Bonferroni’s t test for all post hoc pairwise comparisons. In some cases, homogeneity of regression coefficients was tested with the use of an F test in an analysis of covariance in accordance with the procedures of Steele and Torrie.20 For all statistical testing, P values of less than .05 were considered significant. RESULTS Ion chromatography. A typical conductivity profile for human serum oxalate after ion chromatography, carried out under the conditions established and optimized in preliminary experiments, is presented in Fig 1, A. Although the oxalate anion elutes as distinct peak at about 15 minutes, it is clearly minor in terms of total conductivity and appears on the shoulder(s) of 1 or more other ion species. To validate the oxalate peak derived from serum ultrafiltrates injected onto the column, we adjusted human/rat ultrafiltrate samples to pH 3.2 and treated them with oxalate decarboxylase (20 mU) at 37°C for 60 minutes. This enzyme, which yields stoichiometric quantities of formate and CO2, was prepared from the mycelium of Flammulina velutipes as described by Shimazono and Hayaishi.21 We measured the completeness of this reaction by adding carbon 14 –labeled oxalate (0.4 ␮Ci/mL) to a parallel ultrafiltrate sample and measuring a 50% reduction in counts per minute on liquid-scintillation spectrometry (Beckman LS 6500) after the enzyme reaction and volatilization of labeled CO2. The nonlabeled sample ultrafiltrates, enzyme-treated or not treated (control), were injected onto the column after the removal of chloride, as described above. The results, illustrated in Fig 1, B, for human serum confirm that the peak is caused by oxalate. The same result was obtained for the rat samples (data not shown). Matrix effects and Clⴚ interference. To assess the matrix effect of serum ultrafiltrate and the impact of potentially interfering ions, primarily Cl⫺, we first evaluated the oxalate peak area as a function of oxalate concentration in simple aqueous solutions and in ultrafiltrates of human serum with and without removal of Cl⫺. We initially generated 3 separate standard curves to examine this issue. First, we made aqueous standards by dissolving oxalate in purified water. A second and a third set of standards (internal standards) were made

Fig 1. A typical ion-chromatographic conductivity elution profile of Cl⫺-free human serum ultrafiltrate (A) in which the oxalate eluted at 14.5 minutes, as indicated by the arrow. The oxalate peak was validated by means of enzymatic degradation (B); elution profiles were examined after incubation with oxalate decarboxylase (dashed line) or without enzyme (solid line). Oxalogenesis occurred in the contol sample (solid line) under the incubation conditions required for decarboxylase action, leading to an increase in the area of the oxalate peak, which eluted at 14.6 minutes.

with the use of serum ultrafiltrate with and without Cl⫺ removal as the diluent. We determined the basal oxalate concentration in the ultrafiltrate diluent in advance and subtracted it to generate individual points for the 2 internal standard curves. As shown in Fig 2, the slopes of both standard curves prepared using serum ultrafiltrate (internal standards) were found to be significantly different from each other and from that of the aqueousstandard curve. These results indicate that there are independent background matrix effects of both serum ultrafiltrate and Cl⫺. The matrix effect of Cl⫺ was also reproduced by the addition of increasing amounts of sodium chloride to a 5 ␮mol/L aqueous oxalate stan-

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cantly different from the mean (1.69 ⫾ 0.20 ␮mol/L) obtained with the the internal Cl⫺-free standard curve. Because of this outcome, it can be reasonably argued that the use of internal standards may not be essential and the practicality of applying an aqueous oxalate standard curve can also be justified on the basis of limited sample volumes. Using human serum and Cl⫺free internal standards, we found the interassay and intraassay coefficients of variation to be 2.6% (n ⫽ 6) and 6.6% (n ⫽ 6) respectively. Effects of sample handling on serum oxalate determinations. Deproteinization of serum or plasma samples

Fig 2. The relationship between increasing oxalate concentration and oxalate peak area as determined with the use of ion chromatography in a series of aqueous standards and in 2 series of internal standards, with and without Cl⫺ removal. A regression analysis is presented, with the points representing the means of duplicate measurements. The slopes are significantly different from each other (P ⬍ .005).

dard (data not shown). In summary, the addition of 150 mmol/L NaCl and 171 mmol/L NaCl to the aqueous standard reduced the signal by 48% and 56%, respectively. It is also notable that the addition of 21 mmol/L HCO3⫺ had no effect on the signal. The practical impact of Cl⫺ removal before ionchromatographic analysis of serum oxalate was substantiated in the following manner. We compared oxalate concentrations in serum ultrafiltrate, with and without Cl⫺ removed, using respective ultrafiltrate matrixes (with or without Cl⫺) for internal standardization, in addition to the aqueous-standard curve. As shown in Table I, the oxalate concentration in every serum ultrafiltrate was lower in the presence of chloride, independent of whether an internal or an aqueous standard was used. Because of this Cl⫺ matrix effect, it is clear that chloride removal from serum ultrafiltrate samples is necessary to avoid underestimation of oxalate concentrations. It is also apparent (from Fig 2) that an underestimation of oxalate concentrations is likely to result if an aqueous oxalate standard curve is used to determine oxalate concentrations. However, because of the independent matrix effect of serum ultrafiltrate itself, as mentioned above (and illustrated in Fig 2), the mean serum oxalate concentration of 1.47 ⫾ 0.15 ␮mol/L determined for the 6 Cl⫺-free serum ultrafiltrates (Table I) with the use of the aqueous standards is, in fact, somewhat lower but not signifi-

by means of ultrafiltration is an essential and common step before ion-chromatographic methods for oxalate, yet losses that can accrue during sample processing have not been systematically evaluated. In this series, 12 ultrafiltration devices were compared for both volume and oxalate losses after the ultrafiltration of serum from 6 different individuals. We measured the volume of ultrafiltrate recovered while oxalate recovery was followed by adding carbon 14 –labeled oxalate to serum aliquots and determining the oxalate activity (in counts per minute) in the sample before and after ultrafiltration. As shown in Table II, all filtration devices exhibit some degree of volume and oxalate retention. Loss of volume averaged 61% ⫾ 5% (n ⫽ 12) but ranged from 37% (Amicon Centriflo CF25) to 90% (Millipore Centrifree with acidified serum); tracer loss averaged 68% ⫾ 4% (n ⫽ 12) but ranged from 50% (CLP Apollo) to 89% (Millipore Centrifree with acidified serum). More important, considerable variations in volume and tracer losses were detected among the 6 individual serum samples (and among rat-serum samples) for each of the devices tested. For example, volume and tracer losses ranged from 66% to 90% and 75% to 92%, respectively, when the Millipore Centrifree device was used in the testing of the 6 serum samples. This pattern of variability was observed across all devices, indicating that a general correction factor for volume/tracer losses cannot be applied to precisely calculate individual serum oxalate concentrations. Despite the significant device-dependent volume and tracer losses detailed above, a comparison of the oxalate concentration in the ultrafiltrates when compared with the initial serum sample was particularly informative, as shown in Table II. The device that produced the greatest loss of volume (90%) and tracer oxalate (89%) in the acidified serum (Millipore Centrifree with acidified serum) provided an ultrafiltrate with a mean oxalate concentration just 5% greater than that of the native serum. Ultrafiltrate oxalate concentrations comparable to their respective initial serum concentrations were also derived from the Millipore Microcon-10, Millipore Microcon-30, and Millipore Ultrafree-MC 30

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Table I. Effects of different standardization approaches and Cl⫺ removal on the determination of oxalate in serum from healthy human subjects Serum oxalate concentration (␮mol/L) Serum ID

Internal standard, with Clⴚ

Internal standard, Clⴚ-free

Aqueous standard, normal Clⴚ

Aqueous standard, Clⴚ-free

1.16 0.99 0.58 0.90 0.72 1.72 1.01 ⫾ 0.16*

1.74 2.11 1.36 1.42 1.08 2.40 1.69 ⫾ 0.20†

0.56 0.65 0.37 0.56 0.54 1.03 0.62 ⫾ 0.09‡

1.39 1.81 1.27 1.25 1.07 2.01 1.47 ⫾ 0.15

Female 1 Female 2 Female 3 Male 1 Male 2 Male 3 Mean ⫾ SEM

The oxalate concentrations reported here have been corrected for losses incurred during sample preparation, as described in the text. *Significantly different from Cl⫺-free value produced with internal standardization. † Significantly different from normal Cl⫺ with aqueous standards. ‡ Significantly different from Cl⫺-free with use of aqueous standards.

Table II. Losses of sample volume and oxalate after deproteinization using various ultrafiltration devices

Ultrafiltration device

Amicon Centriflo CF25 CLP Apollo Millipore Amicon Ultra-4 Millipore Centrifree Millipore Centrifree ⫹ HCl Millipore Microcon-10 Millipore Microcon-30 Millipore Ultrafree-MC Millipore Ultrafree-MC Pall Nanosep Sartorius Centrisart I Sartorius Centrisart I

Nominal molecular-weight limit (kD)

Volume applied (mL)

% Volume loss

25 30 30 30 30 10 30 10 30 30 10 20

1.00 1.00 1.00 0.50 0.50 0.20 0.20 0.12 0.12 0.20 1.00 1.00

37 ⫾ 4 40 ⫾ 3 46 ⫾ 4 85 ⫾ 5 90 ⫾ 7 63 ⫾ 4 57 ⫾ 1 56 ⫾ 4 59 ⫾ 6 53 ⫾ 7 75 ⫾ 5 76 ⫾ 4

%

14

C-Ox Loss

58 ⫾ 2 50 ⫾ 2 56 ⫾ 2 88 ⫾ 3 89 ⫾ 2 65 ⫾ 3 60 ⫾ 4 60 ⫾ 3 62 ⫾ 4 60 ⫾ 3 83 ⫾ 2 81 ⫾ 3

% (Oxalate) ultrafiltration/serum

66 ⫾ 3 85 ⫾ 5 82 ⫾ 5 82 ⫾ 5 105 ⫾ 5 98 ⫾ 5 98 ⫾ 6 92 ⫾ 6 97 ⫾ 7 91 ⫾ 6 73 ⫾ 4 74 ⫾ 5

Data presented as the mean ⫾ SEM for 6 human serum samples (3 male, 3 female). Each carbon 14 –labeled sample was processed in duplicate through each device.

(see Table II). Although this is a positive characteristic of the latter devices, a major disadvantage is the requirement for a large initial blood volume for sufficient ultrafiltrate to be recovered for the chromatographic analyses. Further significant losses of oxalate also resulted from binding of oxalate to both the acidified and silver resins used during the preparation of the human samples for ion chromatography. The mean oxalate concentration of ultrafiltrates was reduced by as much as 24% ⫾ 2% (n ⫽ 6) as a result of acid-resin binding and by an additional 6.0% ⫾ 0.8% (n ⫽ 6) as a result of silver-resin binding. Oxalate contamination. We analyzed a few of the more commonly used ultrafiltration devices evaluated above for apparent oxalate contamination of serum ultrafiltrates. The following volumes of purified water were added to the following devices: 1 mL to Apollo, 4

mL to Ultra-4, 1 mL to Centrifree, 500 ␮L to Microcon-10, 500 ␮L to Ultrafree-10, and 1 mL to Centrisart I devices. The water recovered from each ultrafiltration device, analyzed with the use of ion chromatography, revealed an apparent contaminant (oxalate) peak with an elution time similar to that of oxalate. The contribution of the contaminant peak to the calculation of oxalate concentration in each case was 1.6, 0.4 , 2.6, 0.2, 0.1, and 0.7 ␮mol/L, respectively. Consequently, prewashing of the Ultra-4 ultrafiltration devices before use (in the series involving human and rat serum) was routine during this study, and it eliminated the interference. Because the tracer (carbon 14 –labeled oxalate) method employed to evaluate oxalate recovery actually contributes to the total oxalate concentration of the sample, it is possible that this “added oxalate” in turn affects the measurement of tracer recovery. To evaluate

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Fig 3. Recovery of carbon 14 –labeled oxalate from human serum, rat serum, and isotonic calcium– containing buffer (1 mL each) spiked with various amounts of carbon 14 –labeled oxalate (112 ⫻ 103 ␮Ci/mmol) and centrifuged for 30 minutes at 3200g with the use of Amicon Ultra-4 ultrafiltration devices at 4°C. The varying amounts of tracer, from 0.05 to 0.8 ␮Ci, result in oxalate increments ranging from 0.45 to 7.2 ␮mol/L (0.45, 0.9, 1.8, 3.6, and 7.2 ␮mol/L). Data are expressed as mean percentage recovery ⫾SEM of carbon 14 –labeled oxalate after centrifugation of different human or rat serum samples (n ⫽ 3) . The buffer series, which was run in triplicate, contained the following solutes (mmoles per liter): Na⫹, 139.4; K⫹, 5.4; Ca2⫹, 1.2 ; Mg2⫹, 1.2; Cl⫺, 123.2; HCO3⫺, 21.0; H2PO4⫺, 0.6; HPO2⫺, 2.4; 10 glucose; and roughly 2 ␮mol/L oxalate.

this aspect, we measured the recovery of carbon 14 – labeled oxalate as a function of increasing amounts of tracer added to human and rat serum samples, as well as to an artificial buffer with an ionic composition similar to that of mammalian serum. The Amicon Ultra-4 device was used in this series, and 1-mL aliquots of serum or buffer solution were spiked with carbon 14 –labeled oxalate (0.05– 0.8 ␮Ci) corresponding to increases in carrier of 0.45 to 7.2 ␮mol/L. As shown in Fig 3, oxalate recovery was greatest (⬃90%) in the artificial buffer, followed by human (⬃80%) and rat serum (⬃60%). It is also apparent that increasing oxalate activities, resulting from tracer oxalate addition, have no significant effect on the measured oxalate recoveries. Comparison of oxalate concentrations in human and rat serum. To determine whether rat serum oxalate lev-

els are significantly greater than human values, as suggested by the results of previous studies,2,12–14 we compared serum oxalate concentrations between healthy human beings and male Sprague-Dawley rats using identical sample-preparation and analytical techniques, as described above. Amicon Ultra-4 filtration

units were used for rat serum deproteinization, and both volume and tracer recoveries were followed throughout with the use of carbon 14 –labeled oxalate in parallel 1-mL aliquots. The mean oxalate concentration for 6 nonfasted human subjects reported above (Table I) was 1.47 ⫾ 0.15 ␮mol/L, and that for 15 nonfasted male rats was 9.88 ⫾ 0.91 ␮mol/L. As shown in Fig 4, rat serum oxalate levels were not only higher (by about 5 times) but distributed over a wider range in comparison with values measured in human serum. DISCUSSION

Our principal aim in this investigation was to evaluate serum oxalate concentrations in rats because this animal model is the one most commonly used in urolithiasis research.2,14,22–24 Remarkably, relatively little information has been published regarding oxalate levels in the various rat models under normal2,12–14 or experimental conditions,2,12–14,22,23 a circumstance that is most likely a consequence of the limited blood volumes that can be obtained from rodents in anything but a terminal experiment. In the latter type of study, serum oxalate measurements yielded by isotope-dilution and

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enzymatic techniques have indicated that oxalate levels in rat serum may be several times greater than those found in healthy human subjects. Therefore, although the human serum oxalate level is approximately 2 ␮mo/L, according to the current consensus, in healthy male Wistar rats Costello et al12 produced oxalate levels of 5.6 ⫾ 0.6 ␮mol/L with the use of an enzymatic method confirmed by the same authors using an isotopic dilution technique.12 In addition, Hatch et al2,13,14 have measured oxalate concentrations of about 7 ␮mol/L in nonfasting male Sprague-Dawley rats using the same enzymatic method as Costello et al.12 Because of the apparent differences between rats and human beings, the importance of the rat model in studies of calcium oxalate–stone formation, and the necessity for precise knowledge of serum oxalate in the determination of renal oxalate clearance, we have compared serum oxalate levels in healthy rats and human subjects using identical sample handling and ion-chromatography techniques. In establishing the ion-chromatography methodology for our laboratory, it became readily apparent to us that, as for other analytical approaches for oxalate determination, sample handling and preparation are of paramount importance. The chief concerns encountered, beyond prevention of oxalogenesis1,3,8 –11 related to the choice of a standardization method (aqueous vs a serum matrix) and, more significant, the variable degree of oxalate recovery during sample processing (deproteinization and chloride-ion removal). In agreement with the findings of another study,11 we found that serum oxalate determinations are significantly underestimated by ion chromatography in the presence of chloride ion. Although we easily solved this issue by removing the chloride ion before analysis through the use of ionexchange resins, it is possible that the use of a different eluent or chromatography system might also resolve the chloride-ion interference. A correction for oxalate losses during sample deproteinization, before ion chromatography, has not been routinely incorporated in many oxalate methodologies involving chromatographic separation. In addition, ultrafiltrate acidification and chloride removal by means of ion exchange represented other steps in the sample preparation that significantly affected overall oxalate recovery. In this study, we found that all devices used to deproteinize human or rat serum samples retained significant and variable amounts of carbon 14 –labeled oxalate added to monitor the recovery process. Although only small amounts of oxalate are added to the sample in this process, it could be argued that this addition of more oxalate to the serum sample actually depresses the amount of filterable oxalate. However, this was not the case; as shown here (Fig 3), oxalate

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Fig 4. The distribution of human (n ⫽ 6) and rat (n ⫽ 15) serum oxalate concentrations, as measured with the use of ion chromatography. The mean serum oxalate concentration in rats (9.88 ⫾ 0.91 ␮mol/L) was significantly higher than that in human subjects (1.47 ⫾ 0.15 ␮mol/L; P ⬍ .05).

recoveries were independent of added oxalate in human (⬃80%) and rat (⬃60%) serum, as well as artificial serum (⬃90%), for 1 device frequently used in our laboratory. These results also suggest that the less-thanideal recoveries of oxalate during the ultrafiltration process of serum with this particular device are due in part to binding to the filter (⬃10%; ⬃90% was recovered from the protein-free buffer solution) and also binding to serum proteins (⬃10% and ⬃30%, for human and rat serum, respectively, in the study presented in Fig 3). It is notable that although recoveries of tracer oxalate in the ultrafiltrate vary between individuals and between rats, we have also found variable recoveries when blood is drawn at different times from the same individual (eg, fasting and after a meal). We have not specifically tested each filtration device used in this study in terms of inherent binding of tracer, but we speculate that different filters will bind variable amounts of oxalate and that this, combined with variable binding of oxalate to serum proteins, affects recovery of oxalate in the ultrafiltrate. Regardless of the physical basis of the reduced recovery of oxalate in ultrafiltrates, it can clearly affect conclusions regarding oxalate concentrations in serum if appropriate corrections are not made. The results of the direct comparison of rat and human serum oxalate concentrations, with the use of identical sample handling to minimize oxalogenesis1 and analytical detection by ion chromatography, clearly support previous conclusions obtained through the use of enzymatic methods2,12–14—namely, that rat serum oxalate concentrations are several times greater than those in

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human serum. (These differences apply regardless of the application of recovery-correction factors.) Therefore the implicit suspicion that serum oxalate values greater than 2 ␮mol/L in nonhuman mammals are erroneously high is not justified when recoveries during sample preparation are established and precautions are taken to prevent oxalogenesis.12–14 Obviously these differences are trivial in an osmotic sense, but they can significantly affect interpretation of renal handling of oxalate based on renal-clearance studies because serum oxalate concentration appears in the denominator of the standard clearance formula. It is both notable and curious that despite a standard diet and dietary intake, rat serum oxalate concentration ranges widely compared with the concentrations found in human subjects who had varied and unrestricted dietary intakes. The physiological bases for these differences are not apparent at this time, but they may arise from differences in dietary input, degradation by intestinal flora, endogenous production rates, renal transport capacities, allometric effects, or some combination of these factors that determine the steady-state level of a circulating solute. Dr Michael Green kindly provided assistance with statistical issues. We also thank Dave Wranovics and his team of phlebotomists: Audra Campbell, Shelevya Stone, Mary Smith, and Wilemina Blake. Ixion Biotechnology, Inc, provided use of the DIONEX ion chromatograph. REFERENCES

1. Costello J, Landwehr DM. Determination of oxalate concentration in blood. Clin Chem 1988;34:1540 – 4. 2. Hatch M, Freel RW. Renal and intestinal handling of oxalate following oxalate loading in rats. Am J Nephrol 2003;23:18 –26. 3. Hatch M. Oxalate status in stone-formers. Two distinct hyperoxaluric entities. Urol Res 1993;21:55–9. 4. Schwille PO, Manoharan M, Rumenapf G, Wolfel G, Berens H. Oxalate measurement in the picomol range by ion chromatography: values in fasting plasma and urine of controls and patients with idiopathic calcium urolithiasis. J Clin Chem Clin Biochem 1989;27:87–96. 5. Wilson DM, Liedtke RR. Modified enzyme-based colorimetric assay of urinary and plasma oxalate with improved sensitivity and no ascorbate interference: reference values and sample handling procedures. Clin Chem 1991;37:1229 –35. 6. Petrarulo M, Cerelli E, Marangella M, Cosseddu D, Vitale C, Linari F. Assay of plasma oxalate with soluble oxalate oxidase. Clin Chem 1994;40(11 pt 1):2030 – 4. 7. Honow R, Simon A, Hesse A. Interference-free sample preparation for the determination of plasma oxalate analyzed by HPLCER: preliminary results from calcium oxalate stone-formers and non-stone-formers. Clin Chim Acta 2002;318(1-2):19 –24.

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8. Hatch M. Spectrophotometric determination of oxalate in whole blood. Clin Chim Acta 1990;193:199 –202. 9. Rolton HA, McConnell KN, Modi KS, Macdougall AI. A simple, rapid assay for plasma oxalate in uraemic patients using oxalate oxidase, which is free from vitamin C interference. Clin Chim Acta 1989;182:247–54. 10. Kasidas GP, Rose GA. Measurement of plasma oxalate in healthy subjects and in patients with chronic renal failure using immobilised oxalate oxidase. Clin Chim Acta 1986;154:49 –58. 11. Hagen L, Walker VR, Sutton RA. Plasma and urinary oxalate and glycolate in healthy subjects. Clin Chem 1993;39:134 – 8. 12. Costello JF, Smith M, Stolarski C, Sadovnic MJ. Extrarenal clearance of oxalate increases with progression of renal failure in the rat. J Am Soc Nephrol 1992;3:1098 –104. 13. Hatch M, Freel RW, Vaziri ND. Intestinal excretion of oxalate in chronic renal failure. J Am Soc Nephrol 1994;5:1339 – 43. 14. Hatch M, Freel RW. Angiotensin II involvement in adaptive enteric oxalate excretion in rats with chronic renal failure induced by hyperoxaluria. Urol Res 2003;31:426 –32. 15. Skotty DR, Nieman TA. 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. J Chromatogr B Biomed Appl 1995;665:27–36. 16. Marangella M, Petrarulo M, Vitale C, Cosseddu D, Linari F. Plasma and urine glycolate assays for differentiating the hyperoxaluria syndromes. J Urol 1992;148(3 pt 2):986 –9. 17. Petrarulo M, Bianco O, Marangella M, Pellegrino S, Linari F, Mentasti E. Ion chromatographic determination of plasma oxalate in healthy subjects, in patients with chronic renal failure and in cases of hyperoxaluric syndromes. J Chromatogr 1990;511: 223–31. 18. Hoppe B, Kemper MJ, Bokenkamp A, Portale AA, Cohn RA, Langman CB. Plasma calcium oxalate supersaturation in children with primary hyperoxaluria and end-stage renal failure. Kidney Int 1999;56:268 –74. 19. Marangella M, Petrarulo M, Vitale C, Daniele PG, Sammartano S, Cosseddu D, et al. Serum calcium oxalate saturation in patients on maintenance haemodialysis for primary hyperoxaluria or oxalosis-unrelated renal diseases. Clin Sci (Lond) 1991;81: 483–90. 20. Steel RGD, Torrie JH. Principles and procedures of statistics. 2nd ed. New York, NY: Mc Graw-Hill; 1980. 21. Shimazono H, Hayaishi O. Enzymatic decarboxylation of oxalic acid. J Biol Chem 1957;227:151–9. 22. Fan J, Chandhoke PS, Grampsas SA. Role of sex hormones in experimental calcium oxalate nephrolithiasis. J Am Soc Nephrol 1999;10(suppl 14):S376 – 80. 23. Straub B, Muller M, Schrader M, Goessl C, Heicappell R, Miller K. Intestinal and renal handling of oxalate in magnesium-deficient rats. Evaluation of intestinal in vivo 14C-oxalate perfusion. BJU Int 2002;90:312– 6. 24. Khan SR. Animal models of calcium oxalate nephrolithiasis. In: Khan SR, editor. Calcium oxalate in biological systems. Boca Raton, Fla: CRC Press; 1995:343–59.