• View in gallery

    Plasma clearance of inulin (CLinulin) versus the plasma clearance of iohexol (CLiohexol; A) and the difference between CLinulin and CLiohexol versus the mean for CLinulin and CLiohexol (B) in 37 dogs. Each symbol represents results for 1 dog. Values for CLinulin and CLiohexol were standardized on the basis of body weight and calculated by use of the trapezoidal method for 9 plasma samples obtained over a 6-hour period. In panel B, the mean value (solid line) was −0.24 mL/min/kg and the limits of agreement (ie, 2 SD [dotted lines]) were mean ± 1.26 mL/min/kg.

  • View in gallery

    The difference between the plasma clearance of inulin and the plasma clearance of iohexol standardized on the basis of ECFV (ie, GRF/ECFVinulin and GRF/ECFViohexol, respectively) versus the mean for GRF/ECFVinulin and GRF/ECFVinulin in 37 dogs. Each symbol represents results for 1 dog. Values for GRF/ECFVinulin and GRF/ECFViohexol were calculated by use of a 2-compartment model for 9 plasma samples obtained over a 6-hour period. Mean value (solid line) was 3.00 mL/min/kg, and the limits of agreement (ie, 2 SD [dotted lines]) were mean ± 8.88 mL/min/kg.

  • View in gallery

    Values for CLiohexol (A [n = 43 dogs]) or CLinulin (B [35]) determined by use of the reference 9-sample method compared with a 3-sample limited-sample method for plasma samples obtained during the elimination phase. Values were estimated for the 3-sample method via use of a dog-specific equation (white triangles) or the Brøchner-Mortensen formula (black squares); reference CLiohexol and CLinulin were determined for 9 plasma samples (which were obtained during a 6-hour period) by use of a trapezoidal method. Regression lines were calculated for the dogspecific equation and the Brøchner-Mortensen formula.

  • View in gallery

    Values for CLiohexol (A [n = 43 dogs]) or CLinulin (B [35]) determined by use of the reference 9-sample method compared with a 4-sample limited-sample method for plasma samples obtained during the rapid distribution phase and the elimination phase. Values were estimated for the 4-sample method via a 2-comparment method; reference CLiohexol and CLinulin were determined for 9 plasma samples (which were obtained during a 6-hour period) by use of a trapezoidal method. Regression lines were calculated for the prediction of the 9-sample value from the 4-sample value.

  • View in gallery

    The difference between the clearance of iohexol determined by use of the reference 9-sample method (CL9s) and a 2-sample limited-sample method (CL2s) versus the mean of the values for the 9- and 2-sample methods in 18 dogs. Plasma samples for the 9-sample method (which were obtained during a 6-hour period) were analyzed by use of HPLC and UV detection, whereas serum samples for the 2-sample method (which were obtained during the elimination phase) were analyzed by use of x-ray fluorescence. Mean value (solid line) was −0.03 mL/min/kg, and the limits of agreement (ie, 2 SD [dotted lines]) were mean ± 0.80 mL/min/kg.

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Glomerular filtration rate in dogs as estimated via plasma clearance of inulin and iohexol and use of limited-sample methods

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  • 1 Departments of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science, 0454 Oslo, Norway
  • | 2 Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, 0454 Oslo, Norway
  • | 3 Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, 0454 Oslo, Norway
  • | 4 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616
  • | 5 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616

Abstract

Objective—To compare plasma clearance of inulin and iohexol determined by use of 9 plasma samples for evaluation of glomerular filtration rate in dogs and to evaluate limited-sample approaches for evaluation of plasma clearance of these markers.

Animals—43 dogs of various breeds that weighed between 5.5 and 63 kg and that had various degrees of renal function.

Procedures—9 plasma samples were obtained from each dog at 5 minutes to 6 hours after IV bolus injection of iohexol and inulin. Clearance was calculated by use of results for all 9 samples (ie, reference method). Results for 3 limited-sample strategies for determination of plasma clearance of iohexol and inulin were compared with results for the reference method.

Results—Mean clearance of inulin and iohexol for the reference method was 2.72 and 2.48 mL/min/kg, respectively. The mean difference between clearance of these 2 markers for the reference method was 0.24 mL/min/kg. In general, use of the limited-sample strategies yielded clearance values similar to those for the reference method. More accurate estimates of clearance were obtained for iohexol than for inulin by use of the limited-sample methods.

Conclusions and Clinical Relevance—Use of iohexol and inulin yielded similar but not identical results for plasma clearance. Accuracy for limited-sample methods would be acceptable for many clinical and research situations. (Am J Vet Res 2010;71:1100–1107)

Abstract

Objective—To compare plasma clearance of inulin and iohexol determined by use of 9 plasma samples for evaluation of glomerular filtration rate in dogs and to evaluate limited-sample approaches for evaluation of plasma clearance of these markers.

Animals—43 dogs of various breeds that weighed between 5.5 and 63 kg and that had various degrees of renal function.

Procedures—9 plasma samples were obtained from each dog at 5 minutes to 6 hours after IV bolus injection of iohexol and inulin. Clearance was calculated by use of results for all 9 samples (ie, reference method). Results for 3 limited-sample strategies for determination of plasma clearance of iohexol and inulin were compared with results for the reference method.

Results—Mean clearance of inulin and iohexol for the reference method was 2.72 and 2.48 mL/min/kg, respectively. The mean difference between clearance of these 2 markers for the reference method was 0.24 mL/min/kg. In general, use of the limited-sample strategies yielded clearance values similar to those for the reference method. More accurate estimates of clearance were obtained for iohexol than for inulin by use of the limited-sample methods.

Conclusions and Clinical Relevance—Use of iohexol and inulin yielded similar but not identical results for plasma clearance. Accuracy for limited-sample methods would be acceptable for many clinical and research situations. (Am J Vet Res 2010;71:1100–1107)

The GFR is generally regarded as the best overall variable for use in assessing renal function in human and veterinary medicine.1,2 Traditionally, GFR has been estimated by measuring urinary clearance of inulin, which is a fructose polymer. However, methods for measuring urinary clearance of a substrate require accurate collection of urine, which may be cumbersome in animals. Plasma clearance methods are attractive because problems related to collection of urine samples are avoided. However, they have greater potential for error if there is extrarenal clearance of the filtration marker or if the pharmacokinetic model used is not appropriate.3 The extreme variability in the size of domestic dogs has fostered the convention of indexing GFR to body weight or body surface area to establish references. This standardization of clearance to body size remains an unresolved issue in humans4,5 and requires further validation in dogs.6

Inulin is one of the markers used for GFR estimation; however, inulin is expensive and sample handling and laboratory analysis of inulin is cumbersome. Iohexol, a radiographic contrast medium, has been proposed as a marker for GFR and has the advantages that it is readily available, less costly than inulin, and remains stable in plasma, making it feasible to ship samples to a laboratory without the need to include a coolant or the requirement for immediate (ie, overnight) delivery. The ideal GFR marker should be nontoxic; however, a potential for infrequent toxic reactions exists for both inulin and iohexol.

Iohexol appears to fulfill the criteria for a useful filtration marker in most species. It has been validated against reference GFR markers and currently is used extensively in human medicine.1,7 Use of iohexol in dogs has been described in several reports,6,8–16 and iohexol has been compared with other commonly used markers in dogs.

A sensitive laboratory analysis of iohexol is commonly performed by use of HPLC separation and UV determination.17,18 This method is expensive when samples are not analyzed in batches.

A simple x-ray fluorescence–based method by use of an instrumenta specifically designed for measurement of iohexol has been described,19 but that instrument is not currently commercially available. Other laboratory methods for iohexol quantitation have also been described.9,11,13,20,b

Limited-sample strategies for simple estimates of GFR by use of plasma clearance have been evaluated. To define the complete plasma elimination curve after a bolus injection of a marker requires frequent collection of samples during the rapid distribution phase of the curve and the terminal monoexponential elimination phase. This is difficult to achieve in a busy clinical practice or in large-scale research studies. Reducing the number of samples may require the use of correction formulas. A commonly used correction formula, the Brøchner-Mortensen formula, is based on results of studies10,12,21 in adult humans. For dogs, speciesspecific correction formulas have been developed for iohexol.6,12

Among these limited-sample strategies, 2 approaches have been evaluated: samples collected at hourly intervals during the second monoexponential elimination phase of the plasma elimination curve with use of a regression formula to predict the complete curve value,6,12 or collection of samples at intervals during both the rapid distribution phase and the monoexponential phase of the elimination curve.22,23

In a study24 in children, researchers compared results of a 2-sample method in which samples were collected during the elimination phase with results of a 4-sample method in which samples were collected during both the distribution and elimination phase. Similar accuracy was detected whether 2 or 4 samples were used. In dogs, a limited-sample method6 (the 2- and 3-hour 1-compartment method for plasma clearance of iohexol via x-ray fluorescence) has been provided by the veterinary central laboratoryc at the Norwegian School of Veterinary Science since 1996.

The primary objectives of the study reported here were to compare plasma clearance of inulin and iohexol in dogs, to evaluate indexing of GFR to body weight and to the ECFV in dogs of various sizes, to evaluate 3 limited-sample strategies for estimation of GFR, and to compare results for a 2-sample x-ray fluorescence–based method with results for a 9-sample HPLC-based method for iohexol. An additional objective was to define pharmacokinetic parameters for the 2 GFR markers.

Materials and Methods

Dogs—Forty-three client-owned dogs that required estimation of GFR and were admitted to the small animal clinic of the Norwegian School of Veterinary Science between 1999 and 2005 were enrolled in the study. Body weight of the dogs ranged from 5.5 to 63 kg. Of the 43 dogs, 4 had azotemia or proteinuria (or both) and were enrolled for monitoring and prognostic evaluation, 17 had medical conditions with potential renal involvement (ie, unexplained polyuria-polydipsia, hyperadrenocorticism and proteinuria, cardiac disease, urinary tract infection, pyometra, or ectopic ureter with hydronephrosis), and 22 were healthy dogs that were admitted for screening purposes to detect potential heritable renal disease. The 22 healthy dogs comprised 12 Norwegian Elkhounds, 8 Leonbergers, and 2 Boxers; there was a variety of other breeds of various sizes among the other 21 dogs. Each owner provided informed consent for participation of their dog in the study. The study was conducted in accordance with established standards for the care and use of animals in accordance with Norwegian animal welfare legislation.

Plasma clearance procedures—Food was withheld from all dogs for 12 hours before the study, but all dogs had ad libitum access to water. On the day of the study, each dog was weighed and catheters were inserted in both cephalic veins (1 for injection and the other for collection of blood samples). When a cephalic vein could not be used, the ipsilateral saphenous vein was used. Both inulin and iohexol were administered to 37 of 43 dogs; the remaining 6 dogs received only iohexol because of a temporary unavailability of inulin.

Injections of inulin and iohexol were performed sequentially; the order in which the markers were injected was randomized (ie, every other dog received inulin or iohexol first) for the 37 dogs. Both markers were injected during a period of approximately 1 minute; iohexol was usually injected a bit slower than was inulin because of the higher viscosity of iohexol, and the midpoint of the injection of both markers was designated as time 0. Each dog received 40 to 198 mg of inulin/kg from a solution containing 100 mg of inulin/ mLd and 129 to 658 mg of iohexol/kg from a solution containing 647 mg of iohexol/mL (300 mg of iodine/ mL).e The lower dosages were used during the initial phase of the study because of the possibility of toxicosis (3 dogs with mild azotemia were included in the initial phase of the study).

The first milliliter of blood collected from the blood-collection catheter was discarded and then 3 mL of blood was collected into heparinized tubes. The midpoint of each blood collection was recorded as the sample time. After blood collection, the catheter was flushed with 1 mL of saline (0.9% NaCl) solution and then filled with heparinized saline solution.

Blood samples were collected from all 43 dogs immediately before (time 0) and 5, 15, 30, and 60 minutes and 2, 3, 4, 5, and 6 hours after marker injection to provide plasma for analysis of inulin and iohexol concentrations. The exact time of sample collection was used in pharmacokinetic analysis. Plasma samples were divided into equal aliquots and stored at −80°C until analysis. Samples for inulin analysis were transported on dry ice to a university laboratoryf for analysis.

An additional 10 mL of blood was collected from 41 of the 43 dogs before injection and at 2 and 3 hours after injection and placed in serum tubes; these samples were used to provide serum for use in the 2-sample x-ray fluorescence method for analysis of iohexol concentrations.

Analysis of inulin concentrations—Inulin concentrations were measured via an automated inulin assay procedure on an analyzerg by use of an enzymatic method.25 Briefly, inulin was hydrolyzed by use of inulinaseh into single fructose units. The reduction of the generated fructose by nicotinamide adenine dinucleotide dehydrogenase (ie, NADH) to sorbitol was catalyzed by use of sorbitol dehydrogenase.i The reduction of fructose was detected as a decrease in absorbance at 340 nm and was proportional to the concentration of fructose in the sample. Results from unknown samples were compared with values on a standard curve for fructose in 4% bovine serum albumin. A sample of plasma was obtained prior to inulin injection and used to correct for any background interference of fructose in plasma. Control samples for calibration of the assay were prepared from a stock solution of inulin in 4% bovine serum albumin for plasma samples.

For inulin, low concentrations in the last plasma samples impeded calculation of limited-sample clearance values. Two dogs had to be removed from the subsequent limited-sample method comparisons; therefore, values for 35 dogs were compared.

Analysis of iohexol concentrations by use of HPLC—Iohexol concentrations in plasma samples were determined via HPLC by use of a systemj that consisted of a quaternary pump, an online degasser, a thermostat-controlled column, an autosampler, and a UV diode array detector with a 13-µL flow cell and 10-mm path length. Iohexol was separated on a 5-μm analytic reversed-phase column (inner diameter, 4.6 × 250 mm)k by use of a modification of the method described elsewhere.17 The mobile phase was 5% acetonitrile in water with a flow rate of 0.9 mL/min. Total run time was 60 minutes, and the injection volume was 15 μL for all samples. Column temperature was 25°C.

Iohexol consists of 2 stereoisomers, endoiohexol and exoiohexol, with the exoiohexol form yielding the dominant peak. The iohexol concentration was calculated from the height of the exoiohexol absorbance peak at 244 nm by use of a 4-nm slit and a peak width of 0.2 minutes (response time, 4 seconds). For plasma samples, the retention times for the 2 stereoisomers were 7.5 and 8.6 minutes, respectively.

Accuracy was calculated by use of the following equation: ([mean value – standard value]/mean value) × 100. The accuracy for 5 parallel standards in plasma was 5.5% at 1 mg/mL, −2.5% at 10 mg/mL, and 0.3% at 100 mg/mL.

Standards were prepared from a stock solution of 647 mg of iohexol/mL, which corresponded to 300 mg of iodine/mL.d Plasma standards (1, 10, 50, 100, and 250 mg/mL) were made by diluting the stock solution with pooled plasma obtained from 10 healthy dogs from which food was withheld. Protein was removed from samples and standards by the addition of 1 volume of a solution of acetonitrile:ethanol (1:1 [vol:vol]), which were allowed to incubate at 4°C overnight; this was followed by centrifugation at 15,000 × g for 30 minutes. The supernatant was diluted by the addition of 3 volumes of HPLC-grade waterl before injection. All steps were performed with solutions cooled on ice.

Analysis of iohexol by use of x-ray fluorescence—Iohexol concentrations were quantified via analysis of iodine concentration in serum by use of x-ray fluorescence in 3-mL serum samples, as described elsewhere.19 Results for serum samples with < 0.05 mg of iodine/mL were not considered reliable; the detection limit was 0.014 mg of iodine/mL. Pharmacokinetic calculations were performed. The Brochner-Mortensen formula21 in the software for the x-ray fluorescence analyzer was not used. For 23 dogs, the dosage of iohexol was too low to provide adequate serum concentrations by use of this analytic method or an inadequate sample volume was obtained, both of which resulted in the need to dilute the sample into the lower part of the analytic range, which caused loss of accuracy. For these dogs, the generated results were considered unreliable. Results for the 2-sample x-ray fluorescence method were available for comparison in only 18 dogs.

Pharmacokinetic analysis—Total plasma clearance of inulin and iohexol was calculated via a commercially available software programm by use of a noncompartmental (trapezoidal) method for the full curve generated for the 9 plasma samples (reference method).3 Clearance for the reference method was calculated as CLtrap = dose/AUC, with the AUC calculated by use of the complete trapezoidal method. In addition, plasma clearance of inulin and iohexol was calculated via a 1-compartmental method for 3 limited-sample methods.3 For most comparisons, clearance was standardized on the basis of body weight, but standardization on the basis of ECFV was also evaluated, as described elsewhere.6

The estimate for GFR/ECFV was calculated without separate calculation of ECFV by use of the equation5:

article image

where AUMC is the area under the first moment curve.

Three limited-sample methods were evaluated for estimation of GFR. The methods involved use of results obtained by use of HPLC for 3 and 4 of the 9 plasma samples as well as results obtained by use of x-ray fluorescence for 2 serum samples that were obtained at 2 and 3 hours after marker injection.

For the 3-sample method, CLtrap was predicted via an empirical polynomial regression equation for the 1-compartmental method by use of 3 samples (plasma samples obtained at 2, 3, and 4 hours after injection during the elimination phase). The CLtrap was predicted by use of the following empirical dog-specific polynomial regression equation6:

article image

where CL1comp234 is the clearance value obtained by use of the 3 samples.

For the 4-sample method, CLtrap was calculated from the 2-compartmental model values by use of 4 samples (plasma samples obtained at 5 and 15 minutes after injection during the distribution phase and plasma samples obtained at 2 and 3 hours after injection during the elimination phase). The CLtrap was predicted via a spreadsheet programn by use of a 2-compartmental pharmacokinetic method for the following equation:

article image

where C1 and C2 represent the intercept for the distribution phase and elimination phase of the curve, respectively, and λ1and λ2 represent the rate constant for the distribution phase and elimination phase of the curve, respectively. Two time points in each phase were used without any correction formulas.

For the 2-sample method, CLtrap was predicted via an empirical polynomial regression equation for the 1-compartmental method by use of 2 samples (serum samples obtained at 2 and 3 hours after injection during the elimination phase). The CLtrap was predicted by use of the following dog-specific equation for these specific sample collection times6:

article image

where CL1comp23 is the clearance predicted by use of the 2 samples.

Statistical analysis—Results for the 2 markers were compared by evaluating differences against mean plots, with limits of agreement calculated as the difference ± 2 SD.26,27 The comparison was performed for clearance standardized on the basis of body weight and on the basis of ECFV. Values around the mean as well as individual outliers were detected in the mean/difference plot.

Clearance values for the 3- and 4-sample limited-sample methods were compared with the clearance value for the reference method by use of scatterplots and linear regression analysis.26 Statistical analyses were performed by use of a commercially available statistical package.o For the 2-sample limited-sample method, which involved x-ray fluorescence analysis, results were compared via calculation of limits of agreements, as described elsewhere.26

Results

Plasma clearance for the reference method—The difference between plasma clearance of inulin and iohexol versus the mean plasma clearance for inulin and iohexol has been reported elsewhere.p Reference GFR of the 2 markers as estimated via plasma clearance for all 9 samples was similar (Figure 1). Mean plasma clearance of inulin and iohexol was 2.72 and 2.48 mL/ min/kg, respectively. The mean difference between the methods was 0.24 mL/min/kg. The mean plasma clearance for iohexol was 0.24 mL/min/kg higher than the mean plasma clearance for inulin; however, there was variation for which marker yielded the highest result (Figure 1). There was no detectable systematic pattern related to the dosage, size of the dog, or the marker that was injected first to account for the differences between clearance values between markers in individual dogs.

Figure 1—
Figure 1—

Plasma clearance of inulin (CLinulin) versus the plasma clearance of iohexol (CLiohexol; A) and the difference between CLinulin and CLiohexol versus the mean for CLinulin and CLiohexol (B) in 37 dogs. Each symbol represents results for 1 dog. Values for CLinulin and CLiohexol were standardized on the basis of body weight and calculated by use of the trapezoidal method for 9 plasma samples obtained over a 6-hour period. In panel B, the mean value (solid line) was −0.24 mL/min/kg and the limits of agreement (ie, 2 SD [dotted lines]) were mean ± 1.26 mL/min/kg.

Citation: American Journal of Veterinary Research 71, 9; 10.2460/ajvr.71.9.1100

Values for Vdss, MRT, and the elimination half-life were calculated for each marker (Table 1). More than 90% of the AUC was defined after 6 hours in all dogs, except for 3. A mean of 3.7% and 4.9% of the residual AUC was estimated for iohexol and inulin, respectively. In 3 dogs with a very low GFR (0.6 to 0.8 mL/min/kg), 11% to 25% of the AUC was estimated for inulin and 13% to 40% of the AUC was estimated for iohexol. In general, at low concentrations of the markers, the iohexol values more closely followed the computer-estimated curves than did the inulin values.

Table 1—

Mean and range values for pharmacokinetic parameters and the proportion of the total AUC estimated in samples obtained from 37 dogs after bolus IV injections of inulin and iohexol

ParameterInulinIohexol
MeanRangeMeanRange
Clearance (mL/min/kg)2.720.74–5.612.480.64–4.60
Vdss (mL/kg)194131–329221167–357
MRT (min)8245–2568261–147
t1/2 (min)77.852.4–222.474.531.0–180.0
Proportion of AUC estimated (%)3.70.2–25.74.90.5–40.5

t1/2 = Elimination half-life.

Standardization to ECFV—Standardizing clearance estimates to ECFV rather than body weight by use of the reference 9-sample method (ie, GFR/ECFV) yielded an uneven spread of values around the mean difference line (Figure 2). Values for inulin clearance were lower than values for iohexol clearance when renal function was low and higher than values for iohexol clearance when renal function was high. Mean inulin GFR/ECFV was higher than the mean iohexol GFR/ ECFV, which reflected the lower distribution volume for inulin (Table 1).

Figure 2—
Figure 2—

The difference between the plasma clearance of inulin and the plasma clearance of iohexol standardized on the basis of ECFV (ie, GRF/ECFVinulin and GRF/ECFViohexol, respectively) versus the mean for GRF/ECFVinulin and GRF/ECFVinulin in 37 dogs. Each symbol represents results for 1 dog. Values for GRF/ECFVinulin and GRF/ECFViohexol were calculated by use of a 2-compartment model for 9 plasma samples obtained over a 6-hour period. Mean value (solid line) was 3.00 mL/min/kg, and the limits of agreement (ie, 2 SD [dotted lines]) were mean ± 8.88 mL/min/kg.

Citation: American Journal of Veterinary Research 71, 9; 10.2460/ajvr.71.9.1100

3-sample limited-sample method—The relationship between the 3-sample clearance values and the 9-sample reference clearance for iohexol and inulin during the elimination phase was determined by use of the dog-specific equation and the Brøchner-Mortensen formula (Figure 3). Use of the dog-specific equation (R2 = 0.91 for iohexol and R2 = 0.80 for inulin) and the Brøchner-Mortensen formula (R2 = 0.94 for iohexol and R2 = 0.75 for inulin) both yielded similar predictions of the CLtrap values in all dogs. However, use of the Brøchner-Mortensen formula overestimated CLtrap, and an increase in the discrepancy was observed with an increase in GFR.

Figure 3—
Figure 3—

Values for CLiohexol (A [n = 43 dogs]) or CLinulin (B [35]) determined by use of the reference 9-sample method compared with a 3-sample limited-sample method for plasma samples obtained during the elimination phase. Values were estimated for the 3-sample method via use of a dog-specific equation (white triangles) or the Brøchner-Mortensen formula (black squares); reference CLiohexol and CLinulin were determined for 9 plasma samples (which were obtained during a 6-hour period) by use of a trapezoidal method. Regression lines were calculated for the dogspecific equation and the Brøchner-Mortensen formula.

Citation: American Journal of Veterinary Research 71, 9; 10.2460/ajvr.71.9.1100

4-sample limited-sample method—The relationship between clearance values for the 2-phase, 4-sample method and the reference method were determined (Figure 4). The reduction from 9 to 4 samples reduced the accuracy of the clearance estimates (R2 = 0.94 for iohexol and R2 = 0.65 for inulin), compared with the clearance values for the reference method.

Figure 4—
Figure 4—

Values for CLiohexol (A [n = 43 dogs]) or CLinulin (B [35]) determined by use of the reference 9-sample method compared with a 4-sample limited-sample method for plasma samples obtained during the rapid distribution phase and the elimination phase. Values were estimated for the 4-sample method via a 2-comparment method; reference CLiohexol and CLinulin were determined for 9 plasma samples (which were obtained during a 6-hour period) by use of a trapezoidal method. Regression lines were calculated for the prediction of the 9-sample value from the 4-sample value.

Citation: American Journal of Veterinary Research 71, 9; 10.2460/ajvr.71.9.1100

2-sample limited-sample method—In 18 dogs during the elimination phase, calculation by use of 2-sample iohexol clearance yielded a mean difference from the 9-sample reference clearance of −0.03 mL/min/kg. Limits of agreement were 0.80 mL/min/kg (Figure 5).

Figure 5—
Figure 5—

The difference between the clearance of iohexol determined by use of the reference 9-sample method (CL9s) and a 2-sample limited-sample method (CL2s) versus the mean of the values for the 9- and 2-sample methods in 18 dogs. Plasma samples for the 9-sample method (which were obtained during a 6-hour period) were analyzed by use of HPLC and UV detection, whereas serum samples for the 2-sample method (which were obtained during the elimination phase) were analyzed by use of x-ray fluorescence. Mean value (solid line) was −0.03 mL/min/kg, and the limits of agreement (ie, 2 SD [dotted lines]) were mean ± 0.80 mL/min/kg.

Citation: American Journal of Veterinary Research 71, 9; 10.2460/ajvr.71.9.1100

Discussion

Overall, plasma clearances of the 2 markers in the dogs reported here were similar by use of the 9-sample method. Reference ranges for the predicted clearance values determined by use of the limited-sample methods were identical to the reference ranges for clearance determined by use of the reference method; however, accuracy was reduced in various ways by limiting the number of samples. The mean difference in clearance for the reference method between inulin and iohexol was approximately 10%. This was expected because most of the studies1,10,12,23,28–32 in which GFR markers have been compared revealed some discrepancy in the estimated GFR. Reference ranges determined by use of a 3-sample method for a large number of healthy dogs have been reported for plasma clearance of iohexol8 but not for plasma clearance of inulin.

Plasma clearance of a marker molecule for GFR estimation will equal the traditional urinary clearance if the marker is excreted only via glomerular filtration and there is no metabolism, protein binding, tubular secretion, or reabsorption of the marker. In the study reported here, we did not measure urinary clearance, but future studies should focus on measurement of concurrent plasma and urinary clearance. The major advantage of inulin as a marker of glomerular filtration is the vast body of data available from urinary clearance studies and the traditional designation of urinary clearance of inulin as the reference standard for GFR estimation. However, availability and cost of the marker and the need for laboratory analysis of inulin concentrations currently precludes the widespread use of inulin for GFR estimation in clinical or research settings. In addition, it has been suggested33 that not all plasma clearance methods for inulin are equally valid, possibly because of late distribution of inulin into slower equilibrating body compartments. The major advantage of iohexol is its close relationship to inulin with regard to urinary clearance,1,5,10,29 its excretion in the urine in dogs,34 and its stability in plasma and urine, which facilitates shipment of unfrozen samples to laboratories.

The physiologic rationale for indexing GFR to ECFV instead of to body weight is that the regulation of ECFV is, in most cases, closely related to GFR, although this may vary for certain conditions.4,5 Bromide is a marker commonly used to estimate ECFV. Most of the GFR markers distribute in ECFV, and their estimated Vdss values reflect ECFV. Inulin, iohexol, and bromide distribution differ slightly, as exemplified by the differences in Vdss for iohexol and inulin. The range of Vdss was large in the dogs of the present study. This raises a concern about the validity of the pharmacokintetic variable Vdss for use in accurately predicting physiologic ECFV in all individuals. Analysis of Bland-Altman plots revealed that the relationship between plasma clearance of the 2 markers varies with renal function if GFR is standardized to ECFV (Figure 2). This raises further serious questions regarding the validity of this method of standardization in dogs.

Collecting 9 samples over a 6-hour period with short intervals between samples during the first few hours of the collection period may be prohibitive for many investigators performing clearance studies in clinical or research settings. Thus, the quest for valid but simple methods has been ongoing for decades.

The different limited-sample approaches had similar accuracy for the prediction of reference clearance values for each marker, although the R2 values were generally higher for iohexol than for inulin (3- and 4-sample methods; Figures 3 and 4). It is more practical to use samples from only the elimination phase of the curve. Samples from the initial distribution phase, when plasma concentrations change rapidly, need to be collected at precise times to avoid errors in the analysis. The 2-sample method evaluated in this study was as accurate as the 3-sample method, although the use of 3 samples is inherently less vulnerable to error. These observations are in accordance with data reported in dogs6 and supported by results reported for human patients.35 The differences observed when the number of samples was reduced might have been related to the relatively low concentrations of inulin with respect to the limit of detection for the laboratory method. This constraint could be overcome by increasing the dose of inulin administered. However, analysis of some data suggests that there is saturation kinetics at high dosages of inulin, which may preclude the validity of inulin for use as a GFR marker at high dosages because of loss of first-order kinetics.36

Furthermore, the plasma disappearance curves for inulin at all plasma concentrations were not as smooth as were the curves for iohexol. This may partially have been attributable to the inherently larger variation in a biological-enzymatic laboratory method used for inulin, as opposed to the chemical-physical HPLC method used for iohexol. The deviant data points resulted in poorer accuracy of the limited sample methods for inulin than for iohexol.

The clinical validity for use of limited-sample methods was indicated in the study reported here. Among the limited-sample methods evaluated, the 3-sample method for determining iohexol clearance was the single method that optimized both accuracy and practicality as an alternative to the 9-sample method. Reference values for the 3-sample method for determining iohexol clearance in 118 healthy dogs have been published elsewhere.8 In our study, 3 dogs had iohexol clearance values that were clearly lower, and 3 dogs had iohexol clearance values that were slightly higher, than the reference ranges established for the 118 healthy dogs.8 Thus, most of the dogs in the study reported here were considered to have physiologically normal kidney function.

Analysis of data suggests that the period for collection of samples should be extended if kidney function is extremely low when the limited-sample methods are used. Slow elimination of markers requires later time points for sample collection to accurately determine the larger AUC.1,37 In clinical settings, the major indication for GFR estimation may be dogs with relatively good kidney function because an elevated creatinine concentration already is indicative of a low GFR. Optimal times for sample collection in dogs with poor and extremely poor kidney function should be evaluated in future studies.

The study reported here had some limitations. First, we only compared plasma clearance results. Urine collection with calculation of urinary clearances would have provided a better comparison of the reliability of the 2 markers for evaluation of GFR. However, the low percentage of estimated AUC indicated that most of both markers were excreted from the body by 6 hours after injection in dogs with physiologically normal (or near-normal) renal function.

Second, the injected volumes were measured in the syringes, which is less accurate than obtaining a definitive weight of the markers. However, volume registration will most likely be the basis for dosage calculation when these methods are used in clinical settings.

Third, the variability in marker dose was unfortunate. The dose range reported for humans varies from 5 mL/person (independent of body size) up to 2 mL/ kg. The study reported here was conducted over a long time period, and extremely low dosages were used initially because of excessive concerns about toxicosis. The low dosages resulted in many samples with plasma concentrations too low for accurate analysis by use of x-ray fluorescence. Dogs with renal impairment, which may be considered a high-risk group for toxicosis, received the lowest dosages.

Too few healthy dogs of various body sizes and ages were included in the study to enable us to evaluate the potential effect of breed, body size, and age on plasma clearance. The smallest dog in the study (5.5 kg) had high clearance, which is in accordance with the hypothesis that extremely small dogs have a higher GFR, as was reported in another study.8

Iohexol has been used extensively in diagnostic imaging, and there has been considerable focus on its toxic effects. Iohexol is considered to be among the contrast media with the lowest toxicity.38,39 To the authors’ knowledge, there have been no published reports on toxic reactions to iohexol in dogs, possibly because of the relatively small number of dogs exposed to contrast media. In human medicine, the low doses of iohexol used for GFR estimation generally are not considered toxic,38 but patients should be well hydrated before the injection. The use of iohexol and other contrast media is extensive and increasing, whereas the use of inulin for GFR estimation has decreased rapidly during recent years. Toxicosis has also been reported after use of inulin.40

We conclude that plasma clearance of inulin and iohexol is similar but not identical. Accuracy of the limited-sample approaches is comparable, although the inulin clearances for the limited-sample methods deviated more than iohexol clearances did from clearance values determined by use of the reference method. The degree of accuracy needed should be assessed for each specific situation. All limited-sample methods for iohexol clearance may be acceptable in most situations in which available resources do not allow determination of the complete plasma disappearance curve.

ABBREVIATIONS

AUC

Area under the curve

Cltra

Plasma clearance calculated via a trapezoidal method

ECFV

Extracellular fluid volume

GFR

Glomerular filtration rate

HPLC

High-performance liquid chromatography

MRT

Mean residence time

Vdss

Volume of distribution at steady state

a.

The Renalyzer, Provalid, Lund, Sweden.

b.

Bikas V, Magnuson J, Agarwal R, et al. Rapid and accurate determination of non-radioactive iodinated GFR markers (abstr). J Am Soc Nephrol 2007;18:785A.

c.

Heiene R, Moe L. Provetakingsprosedyre for måling av GFR. Available at: www.sentrallaboratoriet.no. Accessed Jan 1, 2000.

d.

Iso-Tex Diagnostics, Friendswood, Tex.

e.

Omnipaque, Nycomed Amersham General Electric, Oslo, Norway.

f.

Renal Research Laboratory, Deptartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, Calif.

g.

Cobas Mira, Roche Diagnostics, Pleasanton, Calif.

h.

Interspex Products, San Mateo, Calif.

i.

Sorbitol dehydrogenase, Roche Molecular Genetics, Palo Alto, Calif.

j.

Series 1100, Agilent Technologies Deutschland GmbH, Waldbronn, Germany.

k.

Spherisorb ODS2, Waters Corp, Milford, Mass.

l.

NANOpure ultrapure water system, Barnstead, van Nuys, Calif.

m.

WinNonlin, version 5.2, Pharsight, Mountain View, Calif.

n.

Excel, Microsoft Norge, Lysaker, Norway.

o.

JMP, SAS Institute Inc, Cary, NC.

p.

Heiene R, Eliassen KA, Risøen U, et al. Plasma clearance of inulin and iohexol over 6 hours in 37 dogs (abstr). J Am Soc Nephrol 2007;18:223.

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Contributor Notes

Supported by a grant from the Norwegian Research Council, and Specific AS, VetXX.

The authors thank Eystein Skjerve for assistance with the statistical analysis.

Address correspondence to Dr. Heiene (reidun.heiene@nvh.no).