- Email: [email protected]
Discordance Between Iothalamate and Iohexol Urinary Clearances
Original Investigation Discordance Between Iothalamate and Iohexol Urinary Clearances Jesse C. Seegmiller, PhD,1 Bradley E. Burns, BA,2 Carrie A. Schinstock, MD,3 John C. Lieske, MD,2,3 and Timothy S. Larson, MD 2,3 Background: Iothalamate and iohexol are contrast agents that have supplanted inulin for the measurement of glomerular filtration rate (GFR) in clinical practice. Previous studies have noted possible differences in renal handling of these 2 agents, but clarity about the differences has been lacking. Study Design: Study of diagnostic test accuracy. Setting & Participants: 150 participants with a wide range of GFRs were studied in an outpatient clinical laboratory facility. Index Tests: Simultaneous urinary clearances of iothalamate, iohexol, and creatinine. Reference Test: None. Outcome: Relative differences between the urinary clearances. Iohexol and iothalamate in plasma and urine were assayed concurrently by a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay. Results: Mean iohexol, iothalamate, and creatinine clearances were 52 6 28 (SD), 60 6 34, and 74 6 40 mL/min/1.73 m2, respectively. The proportional bias of iohexol to iothalamate urinary clearance was 0.85 (95% CI, 0.83-0.88) and was proportional across the GFR range. The mean proportional bias of iohexol clearance compared with creatinine clearance is 1.27 (95% CI, 1.20-1.34), whereas that of iothalamate clearance compared with creatinine clearance is 1.09 (95% CI, 1.03-1.15). Limitations: Lack of reference standard. Conclusions: This study reveals a significant and consistent difference between urinary clearances of iothalamate and iohexol. Comparison of studies reporting renal clearance measurements using iohexol versus iothalamate must account for this observed bias. Am J Kidney Dis. 67(1):49-55. ª 2016 by the National Kidney Foundation, Inc. INDEX WORDS: Glomerular filtration rate (GFR); measured GFR (mGFR); iothalamate; iohexol; exogenous filtration marker; renal clearance; kidney function; liquid chromatography tandem mass spectrometry (LC-MS/ MS); urine; plasma.
Editorial, p. 9
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istorically, inulin has been considered the goldstandard exogenous glomerular filtration rate (GFR) marker, but its routine use is hampered by limited availability for clinical use, high cost, cumbersome administration (intravenous infusion), and analytical issues. Alternative markers for GFR, such as iothalamate and iohexol, have therefore been more widely used. Several studies have suggested that iothalamate1-6 and iohexol7 provide renal clearance measures that are similar to that of inulin. However, only a few studies have directly compared GFR clearance of iothalamate versus iohexol.5,8,9 These reports consistently show that iothalamate clearance is greater than iohexol clearance. Proposed explanations for the differential clearance of these 2 markers include possible tubular secretion of iothalamate,10,11 tubular reabsorption of iohexol,12 or varied volume of distribution13 between the 2 markers. The present study was designed to better understand the differences between the renal clearance of these 2 markers by simultaneously measuring iothalamate and iohexol renal clearance. A newly developed Am J Kidney Dis. 2016;67(1):49-55
liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay allowed concurrent measurement of iothalamate and iohexol in single urine and plasma specimens that minimized both experimental and biological errors.
METHODS Study Design This protocol was approved by the Mayo Clinic Institutional Review Board (no. 09-003297), and signed informed consent was obtained from all study participants as per Mayo Clinic policy. This study was a prospective open-labeled comparative study of 2 index measures for GFR: iothalamate and iohexol.
From the 1Department of Laboratory Medicine, University of Minnesota; 2Department of Laboratory Medicine and Pathology, and 3Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, MN. Received December 2, 2014. Accepted in revised form August 4, 2015. Originally published online October 7, 2015. Address correspondence to Timothy S. Larson, MD, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail: larson.timothy@ mayo.edu 2016 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2015.08.020 49
Seegmiller et al
Setting and Participants Of 1,602 patients who presented to the Mayo Clinic Renal Studies Unit for clinically indicated GFR measurement by their respective ordering provider from October 1, 2009, through February 26, 2010, a total of 150 were enrolled in this study (Fig 1). An attempt was made to enroll equal numbers of patients in 3 tertiles of GFR: ,40, 40 to 80, and .80 mL/min/1.73 m2. Children and adolescents (aged ,18 years) and pregnant women were excluded, as were patients with a known moderate or severe reaction to iodine or contrast agents or those who had an angiographic study in the 12 hours prior to testing. Medical records were abstracted for demographic information, including patient age, sex, and diagnoses. Iothalamate (Conray; iothalamate meglumine injection USP 60%; Mallinckrodt Inc) and iohexol (Omnipaque; iohexol 300 injection; GE Healthcare Inc) were administered by concurrent subcutaneous injection at separate sites on participants’ arms (0.5 mL of contrast 1 0.5 mL of sterile water). Patient preparation and protocols for subsequent timed blood and urine collections were as previously described.14 Briefly, patients were studied in the fasting ($4 hours) state but were encouraged to keep well hydrated with oral intake of water. An equilibration period of 60 minutes following the injection of contrast was allowed, after which urinary clearances of creatinine, iothalamate, and iohexol were determined by urine collection over 45 to 60 minutes and blood sampling at the beginning and end of that period. Ultrasonographic bladder scanning was performed to ensure complete bladder emptying after all urine collections. Bladder catheterization was performed if residual urine was detected (.10 mL or .10% of voided volume). Blood and urine samples were stored refrigerated at 4 C until LCMS/MS analysis. Stability studies confirmed that blood and urine iothalamate and iohexol were stable up to 7 days when stored ambient (20 C), refrigerated (4 C), or frozen (220 C) prior to iothalamate/ iohexol analysis. Measured GFR was calculated as previously described.14,15 Creatinine was measured in blood and urine samples using the Roche Creatinine plus enzymatic assay performed on a cobas c501 per manufacturer’s instructions (Roche Diagnostics).
Liquid Chromatography–Tandem Mass Spectrometry Quantification of iothalamate and iohexol in urine and plasma was performed using a tandem mass spectrometric method developed in the Mayo Clinic Renal Function Laboratory.
Sample Preparation Plasma samples were prepared by pooling 50 mL each of P1 and P2 (plasma drawn at the beginning and end of the clearance study,
respectively) and adding 20 mL of internal standard (d3-iothalamic acid [38 mg/dL] and d3-iohexol [38 mL/dL] prepared in the Mayo Clinic Chemical Synthesis Core Facility, Jacksonville, FL. Samples were deproteinized by the addition of 100 mL of acetonitrile (Sigma Aldrich) followed by vortex mixing (5 seconds) and centrifugation (650g for 5 minutes). A 20-mL aliquot of the resulting supernatant was diluted in 1 mL of water from a NANOpure DIamond ultra pure water system (Barnstead/Thermolyne Corp). Urine samples (U1) were diluted 1:10 with water (100 mL final volume) followed by the addition of the internal standard (20 mL). Samples were deproteinized by the addition of acetonitrile (100 mL), vortex mixed (5 seconds), and centrifuged (650g for 5 minutes). A 20mL aliquot of the resulting supernatant was diluted with 1 mL of water.
Analysis Complete LC-MS/MS method parameter details can be found in Item S1 (provided as online supplementary material). Data in this manuscript are derived from the iothalamate m/z 614.7-455.8 multiple reaction monitoring (MRM) and the iohexol m/z 821.8-602.8 MRM transitions (Fig S1).
Calibration and Control Material The LC-MS/MS method was calibrated using 0.76, 1.52, 7.59, 15.2, 37.9, and 75.9 mg/L of iothalamate from a Conray stock and 1, 2, 10, 20, 50, and 100 mg/L of iohexol from an Omnipaque stock, both diluted in water. Controls were made by the addition of Conray and Omnipaque to plasma and urine samples that did not contain either iothalamate or iohexol. One plasma and 3 urine controls were made to represent 3 GFR categories (low, medium, and high).
In Vitro Dialysis Experiments In order to assess the ultrafilterability of iohexol and iothalamate, in vitro dialysis experiments were performed on 0.5-mL aliquots of waste plasma to which iohexol and iothalamate were added. Slide-A-Lyzer (ThermoScientific) dialysis cassettes with a 2,000-Da molecular weight cutoff were initially hydrated for 2 minutes in dialysate buffer, 0.01 mol/L of phosphate-buffered saline (PBS), containing 0.138 mol/L of sodium chloride, and 0.0027 mol/L of potassium chloride (Sigma Aldrich); loaded with 0.5-mL plasma aliquots; and dialyzed overnight versus PBS dialysate buffer (40 mL) at room temperature under gentle agitation using a titer plate shaker (180 rotations per minute).
Statistical Analysis Clearance data were analyzed by Passing-Bablok regression analysis for method comparison. The slope and y-intercept were calculated along with their 95% confidence intervals (CIs). Relative differences were illustrated by Bland-Altman plots. The relative difference (bias) was further analyzed by application of paired t test. In vitro dialysis experimental data were analyzed by paired t test. General linear regression methods were used to assess the relationships between sex and age with the ratio of iohexol to iothalamate clearances (implemented in SAS, version 9.4 [SAS Institute Inc]). Significance was defined as P , 0.05. Microsoft Excel and Analyse-it for Microsoft Excel, version 2.26 (Analyse-it Software Ltd), were used for all other data analysis. Statistical methods for the analytic validation studies are reported in Item S1.
RESULTS Figure 1. Study participant enrollment. Concurrent urinary clearances of iothalamate, iohexol, and creatinine were obtained in the 150 enrolled study participants (see text for additional enrollment details). 50
The LC-MS/MS assay performance for measuring iohexol and iothalamate in plasma and urine was excellent, with coefficients of variation , 3% and Am J Kidney Dis. 2016;67(1):49-55
Iothalamate and Iohexol Urinary Clearances
recoveries generally .99% across all concentration ranges. Full details of the assay performance are provided in Item S1. Figure 1 summarizes the enrollment of study participants for concurrent measurement of urinary clearances of iothalamate, iohexol, and creatinine. Clinical characteristics of the study population are included in Table 1. More men (n 5 81) than women (n 5 69) were enrolled in the study. Mean age was 54 6 13 (range, 22-81) years. Forty percent (n 5 60) of study participants had previously undergone solid-organ or bone marrow transplantation. Indications for testing in the rest included potential kidney donor evaluation, pre–liver or pre–bone marrow transplantation evaluation, and chronic kidney disease (CKD) staging. Iohexol versus iothalamate clearances are compared in Fig 2A, whereas Figure 2B and C shows creatinine versus iothalamate and iohexol clearances, respectively, for all participants enrolled in the study. Passing-Bablok regression was used to assess these relationships because it is a robust form of regression that places less weight on outlier observations. Of note, the association between iohexol and iothalamate clearances had significant proportional bias (slope 5 0.85; 95% CI, 0.83-0.88), but not a significant constant bias (intercept 5 0.44; 95% CI, 0.43-1.50; Fig 2A). The proportional bias for iothalamate clearances compared with creatinine clearances was significantly less than for iohexol, although both were statistically Table 1. Study Population Characteristics Parameter
No. of participants Age, y Sex, M:F Race White African American Other
Value
150 54 6 13 (22-81) 81:69 91% 1% 8%
Diagnoses Diabetes mellitus Kidney transplant recipient Other transplant recipient Primary glomerular disease Polycystic kidney disease Pre– or post–kidney donor Amyloidosis/multiple myeloma Other
16 (11) 39 (26) 34 (23) 7 (5) 5 (3) 11 (7) 8 (5) 30 (20)
Clearance, mL/min/1.73 m2 Iothalamate Iohexol Creatinine
60 6 34 52 6 29 74 6 40
Note: Unless otherwise indicated, values for categorical variables are given as number (percentage); for continuous variables, as mean 6 standard deviation or mean 6 standard deviation (range). Am J Kidney Dis. 2016;67(1):49-55
significant (slopes of 1.09 [95% CI, 1.03-1.15] and 1.27 [95% CI, 1.20-1.34], respectively). In addition, the constant biases for both iothalamate and iohexol clearances compared with creatinine clearances were both statistically significant (intercepts of 5.27 [95% CI, 2.66-8.60] and 4.63 [95% CI, 1.78-7.68], respectively), resulting in both iohexol and iothalamate clearances being less than creatinine clearance, as expected (Figs 2B and C). Iothalamate and iohexol concentrations were comparable to each other in both plasma and urine (Table 2). By Bland-Altman plot, iothalamate clearance displayed an average 15% bias (P , 0.001) compared to iohexol (Fig 3A). This consistent bias was observed throughout the range of GFRs evaluated. The average bias of creatinine versus iothalamate was 24% (Fig 3B), whereas for creatinine versus iohexol, it was 38% (Fig 3C). To further investigate the consistent 15% difference between renal clearance of the 2 agents, iothalamate and iohexol were added to 10 clinical laboratory plasma waste samples and dialyzed against PBS. The fraction of iohexol in the plasma after versus before overnight dialysis was significantly higher than for iothalamate (0.19 6 0.03 vs 0.11 6 0.02, respectively; P , 0.001; Table 3). These data suggest that iohexol is not as ultrafilterable across a 2,000-Da membrane as iothalamate, perhaps due to differences in protein binding or other as yet undefined differences in diffusibility. Although the ratio of iohexol to iothalamate urinary clearance was not associated with sex (P 5 0.8), it was statistically significantly associated with age (P 5 0.03), but not necessarily clinically associated with age, as evidenced by the very small coefficient of determination (R2 5 0.03).
DISCUSSION The most notable finding of this study is the unequivocal demonstration that urinary clearance of iohexol is on average 15% less than that of iothalamate across a wide range of GFRs. Furthermore, iohexol is less ultrafiltrable than iothalamate across a 2,000-Da molecular weight dialysis membrane in vitro, which likely contributes to their differential clearance by the kidney in vivo. These findings have important implications for the comparison of renal clearances obtained using these 2 methods or when pooling them for larger meta-analyses. In the current study, we developed a new LC-MS/ MS assay to simultaneously analyze iothalamate and iohexol in urine and plasma specimens. This complements our previous work on the development and validation of a similar LC-MS/MS assay for iothalamate alone.15 In developing the LC-MS/MS assay for concurrently measuring iothalamate and iohexol in urine and plasma, minor methodologic changes were 51
Seegmiller et al
Figure 2. Passing-Bablok regression comparison plots of concurrent urinary clearances of (A) iohexol versus iothalamate, (B) creatinine versus iothalamate, and (C) creatinine versus iohexol. The equations for the regression lines (with 95% confidence intervals (CIs) of slope and intercept, respectively, in parentheses) are (A) y 5 0.85x 1 0.44 (0.83 to 0.88; 20.43 to 1.50); (B) y 5 1.09x 1 5.27 (1.03 to 1.15; 2.66 to 8.60); and (C) y 5 1.27x 1 4.63 (1.20 to 1.34; 1.78 to 7.68). Solid black lines are Passing-Bablok regression; dashed lines are 95% CIs of the regression, and thin gray lines are lines of identity, x 5 y. Abbreviations: CreatClr, creatinine urinary clearance; IohexClr, iohexol urinary clearance; IothClr, iothalamate urinary clearance. 52
implemented to the original iothalamate analytical method that slightly improved the performance of the previously reported iothalamate assay; the end result is excellent analytical sensitivity and specificity. Previously our laboratory and others have used capillary electrophoresis to measure iothalamate and/ or iohexol. However, the major advantage of LC-MS/ MS over capillary electrophoresis is greater analytic specificity and hence less susceptibility to interfering substances. A major objective of the current study was to better delineate possible differences between the renal clearances of iothalamate and iohexol. Few studies have directly compared the renal clearances of iothalamate and iohexol. Moreover, typically iohexol clearances are measured by way of plasma clearance, whereas iothalamate is more commonly used with urinary clearance methods. Only one other study directly compared simultaneous renal clearance of iothalamate and iohexol.5 In this small study performed with 14 patients and 3 healthy volunteers, Isaka et al5 reported that the slope of iothalamate versus inulin clearance was 1.06 6 0.02, whereas that for iohexol versus inulin clearance was 0.83 6 0.02. Soveri et al16 recently reviewed prior published reports of the accuracy of GFR measurement methods using renal inulin clearance as reference. Although none of the reported studies simultaneously compared iothalamate, iohexol, and inulin, they noted a mean bias for urinary clearance of iohexol versus inulin of 218 at GFR of 30, 211 at GFR of 60, and 22 at GFR of 90 mL/min/1.73 m2, with slightly positive mean bias of iothalamate across this range of GFRs (17, 16, and 15 mL/min/1.73 m2, respectively). The reported overall mean bias was 211 for iohexol and 16 for iothalamate, a difference of 17 mL/min/ 1.73 m2 between the 2 and thus not too dissimilar to the results of the current study. Interestingly, there were more studies for renal clearance of iothalamate (n 5 13) than iohexol (n 5 2), whereas the reverse was true for plasma disappearance (n 5 1 vs n 5 5, respectively). The authors also cautioned that many of the studies were small. However, the pooled results support the direction of the bias between the 2 methods that we observed here, with iohexol urinary clearance being less than iothalamate urinary clearance. The explanation for the observed difference between iothalamate and iohexol urinary clearance is not certain. Ideally, inclusion of inulin clearance as the goldstandard clearance marker would have been helpful. However, at the time of this study, inulin was not available as an approved injectable substance for humans. Thus, we cannot be certain whether urinary clearance of iothalamate or iohexol is a more accurate representation of GFR. It is interesting to note that early works by Maher et al3 and Sigman et al1 demonstrated Am J Kidney Dis. 2016;67(1):49-55
Iothalamate and Iohexol Urinary Clearances Table 2. Ratios of Urine-Plasma Concentrations for Iothalamate, Iohexol, and Creatinine Marker
Mean 6 SD
Iothalamate Iohexol Creatinine
1.31 6 1.13 1.12 6 0.93 1.76 6 1.71
Abbreviation: SD, standard deviation.
that urinary clearance of inulin and radiolabeled iothalamate were nearly identical across a wide range of GFRs, as did the more recent study of Isaka et al.5 The current study included creatinine clearance as a potential comparator. The bias between iothalamate and creatinine clearance in the current study (24%) is exactly that observed between inulin and creatinine clearance in the study by Isaka et al,5 whereas in the current study, the bias between iohexol and creatinine clearance is much greater (creatinine clearance 38% greater than iohexol). These observations suggest that iothalamate clearance may be closer to measured inulin clearance than iohexol clearance. The current study also suggests that decreased ultrafilterability of iohexol compared to iothalamate, perhaps due to increased plasma protein binding, could account for its relatively lower clearance, a possibility that has been previously discounted by others.8,17 Of note, clearances for both markers have been observed to be higher when measured by plasma disappearance as compared with urinary clearance.8 This also has been the case for inulin18,19 and has previously been attributed to ongoing extrarenal clearance of the markers in the periphery. Future studies comparing renal clearances of iothalamate, iohexol, and inulin, when possible, would be enlightening. A difference in the absorption rate of the 2 analytes from the subcutaneous depot could also potentially result in a difference in clearance results of the 2 analytes. Because the mean concentration from a pooled pre- and postclearance plasma sample was used as the plasma concentration for each clearance measurement, we cannot be certain that the mean value is the most accurate representation of the plasma concentration during the urine collection period for both analytes. Although not a focus of this study, the comparison of creatinine clearances with both iothalamate and iohexol clearances showed, as expected, that creatinine clearances were greater than those of both of the other 2 markers. This is in keeping with multiple other reports, including the recent analysis by Soveri et al.16 Because only relatively recently have standardized creatinine assays been implemented, the older reports may affect direct comparability with the current study. In addition to the lack of availability of the reference marker inulin to assess accuracy, use of a single urine collection of approximately one hour duration for the Am J Kidney Dis. 2016;67(1):49-55
Figure 3. Relative difference (Bland-Altman) plots of concurrent clearances of (A) iohexol versus iothalamate, (B) creatinine versus iothalamate, and (C) creatinine versus iohexol. The x-axis is the average of the 2 respective clearances, the y-axis is the difference of the 2 clearance measurements divided by the average (adjusts for increasing dispersion of measurements with increasing glomerular filtration rate). Solid black lines represent the mean relative difference (%); dashed lines, the 95% confidence limits of agreement; and thin gray lines, present bias 5 0. Abbreviations: CreatClr, creatinine urinary clearance; IohexClr, iohexol urinary clearance; IothClr, iothalamate urinary clearance.
53
Seegmiller et al Table 3. Fraction of Iothalamate and Iohexol Remaining in Plasma Following Overnight In Vitro Dialysis Experiments Marker
Mean 6 SD
Pa
Iothalamate Iohexol
0.11 6 0.02 0.19 6 0.03
,0.001
Abbreviation: SD, standard deviation. By paired t test.
a
clearance measurements is a potential weakness. As such, precise GFR measurement would be strengthened with longer or multiple urine collection periods. However, the goal of the current study was not accurate determination of GFR per se, but rather comparison between the 2 commonly used index GFR methods (iothalamate and iohexol). Simultaneous measurement of both iothalamate and iohexol in the same urine and plasma aliquots controlled for most preanalytic issues such as timing of collections, bladder emptying, and sample processing and thus minimized the influence of those variables in the comparison of the 2 analytes. Regardless of their relative bias compared to inulin clearance, the current study has obvious relevance for comparing data in clinical studies in which GFR was assessed by renal clearance of different markers. Because of the difficulty performing renal clearances from large cohorts, pre-existing clinical data are often pooled, often including various GFR methodologies. Consideration must be given to the potential implications of using pooled data across differing GFR measurement methodologies. For example, to develop the recent CKD-EPI (CKD Epidemiology Collaboration) equations, GFR was measured using iothalamate in 82%, iohexol in 9%, and EDTA in 9%.20 However, most studies using iohexol for GFR measurement rely on plasma clearance measurements rather than urinary clearance, the latter of which was the comparison in the present study. Soveri et al16 noted a mean bias of 27 for plasma clearance versus 12 mL/min/1.73 m2 for urinary clearance of iohexol in relation to inulin clearance, although the number of patients studied in the latter group was limited. Thus, whether plasma versus urinary clearance of iohexol exhibits similar bias relative to iothalamate is unclear and worthy of further evaluation. In conclusion, we show that iohexol renal clearances are lower than iothalamate renal clearances. Decreased ultrafilterability of iohexol, perhaps due to greater protein binding, might contribute to the lower measurements.
ACKNOWLEDGEMENTS Support: None. Financial Disclosure: The authors declare that they have no relevant financial interests. Contributions: Research idea and study design: JCS, TSL; data acquisition: JCS, BEB, CAS; data analysis/interpretation: JCS, 54
JCL, TSL; supervision: TSL. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. TSL takes responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.
SUPPLEMENTARY MATERIAL Figure S1: Representative chromatogram of an iothalamate/ iohexol calibrator. Item S1: Supplemental tandem mass spectrometry methodology for measurement of iohexol. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2015.08.020) is available at www. ajkd.org
REFERENCES 1. Sigman EM, Elwood CM, Knox F. The measurement of glomerular filtration rate in man with sodium iothalamate 131-I (Conray). J Nucl Med. 1965;7:60-68. 2. Elwood CM, Sigman EM, Treger C. The measurement of glomerular filtration rate with 125I-sodium iothalamate (Conray). Br J Radiol. 1967;40:581-583. 3. Maher FT, Nolan NG, Elveback LR. Comparison of simultaneous clearances of 125-I-labeled sodium iothalamate (Glofil) and of inulin. Mayo Clin Proc. 1971;46:690-691. 4. Milutinovic J, Cutler RE, Hoover P, Meijsen B, Scribner BH. Measurement of residual glomerular filtration rate in the patient receiving repetitive hemodialysis. Kidney Int. 1975;8:185-190. 5. Isaka Y, Yamamoto S, Fujiwara Y, et al. [A new analytical method for simultaneous measurement of iothalamate and iohexol]. Rinsho Byori. 1992;40:703-707. 6. Maher FT, Tauxe WN. Renal clearance in man of pharmaceuticals containing radioactive iodine. JAMA. 1969;207:97-104. 7. Brown SC, O’Reilly PH. Iohexol clearance for the determination of glomerular filtration rate in clinical practice: evidence for a new gold standard. J Urol. 1991;146:675-679. 8. Back SE, Krutzen E, Nilsson-Ehle P. Contrast media as markers for glomerular filtration: a pharmacokinetic comparison of four agents. Scand J Clin Lab Invest. 1988;48:247-253. 9. Back SE, Krutzen E, Nilsson-Ehle P. Contrast media and glomerular filtration: dose dependence of clearance for three agents. J Pharm Sci. 1988;77:765-767. 10. Odlind B, Hallgren R, Sohtell M, Lindstrom B. Is 125I iothalamate an ideal marker for glomerular filtration? Kidney Int. 1985;27:9-16. 11. Zurth C. Mechanism of renal excretion of various x-ray contrast materials in rabbits. Invest Radiol. 1984;19:110-115. 12. Masereeuw R, Moons MM, Smits P, Russel FG. Glomerular filtration and saturable absorption of iohexol in the rat isolated perfused kidney. Br J Pharmacol. 1996;119:57-64. 13. Olsson B, Aulie A, Sveen K, Andrew E. Human pharmacokinetics of iohexol. A new nonionic contrast medium. Invest Radiol. 1983;18:177-182. 14. Wilson DM, Bergert JH, Larson TS, Liedtke RR. GFR determined by nonradiolabeled iothalamate using capillary electrophoresis. Am J Kidney Dis. 1997;30:646-652. 15. Seegmiller JC, Burns BE, Fauq AH, Mukhtar N, Lieske JC, Larson TS. Iothalamate quantification by tandem mass spectrometry to measure glomerular filtration rate. Clin Chem. 2010;56: 568-574. Am J Kidney Dis. 2016;67(1):49-55
Iothalamate and Iohexol Urinary Clearances 16. Soveri I, Berg UB, Bjork J, et al. Measuring GFR: a systematic review. Am J Kidney Dis. 2014;64:411-424. 17. Krutzen E, Back SE, Nilsson-Ehle I, Nilsson-Ehle P. Plasma clearance of a new contrast agent, iohexol: a method for the assessment of glomerular filtration rate. J Lab Clin Med. 1984;104:955-961. 18. Florijn KW, Barendregt JN, Lentjes EG, et al. Glomerular filtration rate measurement by “single-shot” injection of inulin. Kidney Int. 1994;46:252-259.
Am J Kidney Dis. 2016;67(1):49-55
19. Rehling M, Moller ML, Thamdrup B, Lund JO, TrapJensen J. Simultaneous measurement of renal clearance and plasma clearance of 99mTc-labelled diethylenetriaminepentaacetate, 51Cr-labelled ethylenediaminetetra-acetate and inulin in man. Clin Sci (Lond). 1984;66:613-619. 20. Inker LA, Schmid CA, Tighiouart H, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med. 2012;367:20-29.
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Editorial, p. 9
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istorically, inulin has been considered the goldstandard exogenous glomerular filtration rate (GFR) marker, but its routine use is hampered by limited availability for clinical use, high cost, cumbersome administration (intravenous infusion), and analytical issues. Alternative markers for GFR, such as iothalamate and iohexol, have therefore been more widely used. Several studies have suggested that iothalamate1-6 and iohexol7 provide renal clearance measures that are similar to that of inulin. However, only a few studies have directly compared GFR clearance of iothalamate versus iohexol.5,8,9 These reports consistently show that iothalamate clearance is greater than iohexol clearance. Proposed explanations for the differential clearance of these 2 markers include possible tubular secretion of iothalamate,10,11 tubular reabsorption of iohexol,12 or varied volume of distribution13 between the 2 markers. The present study was designed to better understand the differences between the renal clearance of these 2 markers by simultaneously measuring iothalamate and iohexol renal clearance. A newly developed Am J Kidney Dis. 2016;67(1):49-55
liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay allowed concurrent measurement of iothalamate and iohexol in single urine and plasma specimens that minimized both experimental and biological errors.
METHODS Study Design This protocol was approved by the Mayo Clinic Institutional Review Board (no. 09-003297), and signed informed consent was obtained from all study participants as per Mayo Clinic policy. This study was a prospective open-labeled comparative study of 2 index measures for GFR: iothalamate and iohexol.
From the 1Department of Laboratory Medicine, University of Minnesota; 2Department of Laboratory Medicine and Pathology, and 3Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, MN. Received December 2, 2014. Accepted in revised form August 4, 2015. Originally published online October 7, 2015. Address correspondence to Timothy S. Larson, MD, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail: larson.timothy@ mayo.edu 2016 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2015.08.020 49
Seegmiller et al
Setting and Participants Of 1,602 patients who presented to the Mayo Clinic Renal Studies Unit for clinically indicated GFR measurement by their respective ordering provider from October 1, 2009, through February 26, 2010, a total of 150 were enrolled in this study (Fig 1). An attempt was made to enroll equal numbers of patients in 3 tertiles of GFR: ,40, 40 to 80, and .80 mL/min/1.73 m2. Children and adolescents (aged ,18 years) and pregnant women were excluded, as were patients with a known moderate or severe reaction to iodine or contrast agents or those who had an angiographic study in the 12 hours prior to testing. Medical records were abstracted for demographic information, including patient age, sex, and diagnoses. Iothalamate (Conray; iothalamate meglumine injection USP 60%; Mallinckrodt Inc) and iohexol (Omnipaque; iohexol 300 injection; GE Healthcare Inc) were administered by concurrent subcutaneous injection at separate sites on participants’ arms (0.5 mL of contrast 1 0.5 mL of sterile water). Patient preparation and protocols for subsequent timed blood and urine collections were as previously described.14 Briefly, patients were studied in the fasting ($4 hours) state but were encouraged to keep well hydrated with oral intake of water. An equilibration period of 60 minutes following the injection of contrast was allowed, after which urinary clearances of creatinine, iothalamate, and iohexol were determined by urine collection over 45 to 60 minutes and blood sampling at the beginning and end of that period. Ultrasonographic bladder scanning was performed to ensure complete bladder emptying after all urine collections. Bladder catheterization was performed if residual urine was detected (.10 mL or .10% of voided volume). Blood and urine samples were stored refrigerated at 4 C until LCMS/MS analysis. Stability studies confirmed that blood and urine iothalamate and iohexol were stable up to 7 days when stored ambient (20 C), refrigerated (4 C), or frozen (220 C) prior to iothalamate/ iohexol analysis. Measured GFR was calculated as previously described.14,15 Creatinine was measured in blood and urine samples using the Roche Creatinine plus enzymatic assay performed on a cobas c501 per manufacturer’s instructions (Roche Diagnostics).
Liquid Chromatography–Tandem Mass Spectrometry Quantification of iothalamate and iohexol in urine and plasma was performed using a tandem mass spectrometric method developed in the Mayo Clinic Renal Function Laboratory.
Sample Preparation Plasma samples were prepared by pooling 50 mL each of P1 and P2 (plasma drawn at the beginning and end of the clearance study,
respectively) and adding 20 mL of internal standard (d3-iothalamic acid [38 mg/dL] and d3-iohexol [38 mL/dL] prepared in the Mayo Clinic Chemical Synthesis Core Facility, Jacksonville, FL. Samples were deproteinized by the addition of 100 mL of acetonitrile (Sigma Aldrich) followed by vortex mixing (5 seconds) and centrifugation (650g for 5 minutes). A 20-mL aliquot of the resulting supernatant was diluted in 1 mL of water from a NANOpure DIamond ultra pure water system (Barnstead/Thermolyne Corp). Urine samples (U1) were diluted 1:10 with water (100 mL final volume) followed by the addition of the internal standard (20 mL). Samples were deproteinized by the addition of acetonitrile (100 mL), vortex mixed (5 seconds), and centrifuged (650g for 5 minutes). A 20mL aliquot of the resulting supernatant was diluted with 1 mL of water.
Analysis Complete LC-MS/MS method parameter details can be found in Item S1 (provided as online supplementary material). Data in this manuscript are derived from the iothalamate m/z 614.7-455.8 multiple reaction monitoring (MRM) and the iohexol m/z 821.8-602.8 MRM transitions (Fig S1).
Calibration and Control Material The LC-MS/MS method was calibrated using 0.76, 1.52, 7.59, 15.2, 37.9, and 75.9 mg/L of iothalamate from a Conray stock and 1, 2, 10, 20, 50, and 100 mg/L of iohexol from an Omnipaque stock, both diluted in water. Controls were made by the addition of Conray and Omnipaque to plasma and urine samples that did not contain either iothalamate or iohexol. One plasma and 3 urine controls were made to represent 3 GFR categories (low, medium, and high).
In Vitro Dialysis Experiments In order to assess the ultrafilterability of iohexol and iothalamate, in vitro dialysis experiments were performed on 0.5-mL aliquots of waste plasma to which iohexol and iothalamate were added. Slide-A-Lyzer (ThermoScientific) dialysis cassettes with a 2,000-Da molecular weight cutoff were initially hydrated for 2 minutes in dialysate buffer, 0.01 mol/L of phosphate-buffered saline (PBS), containing 0.138 mol/L of sodium chloride, and 0.0027 mol/L of potassium chloride (Sigma Aldrich); loaded with 0.5-mL plasma aliquots; and dialyzed overnight versus PBS dialysate buffer (40 mL) at room temperature under gentle agitation using a titer plate shaker (180 rotations per minute).
Statistical Analysis Clearance data were analyzed by Passing-Bablok regression analysis for method comparison. The slope and y-intercept were calculated along with their 95% confidence intervals (CIs). Relative differences were illustrated by Bland-Altman plots. The relative difference (bias) was further analyzed by application of paired t test. In vitro dialysis experimental data were analyzed by paired t test. General linear regression methods were used to assess the relationships between sex and age with the ratio of iohexol to iothalamate clearances (implemented in SAS, version 9.4 [SAS Institute Inc]). Significance was defined as P , 0.05. Microsoft Excel and Analyse-it for Microsoft Excel, version 2.26 (Analyse-it Software Ltd), were used for all other data analysis. Statistical methods for the analytic validation studies are reported in Item S1.
RESULTS Figure 1. Study participant enrollment. Concurrent urinary clearances of iothalamate, iohexol, and creatinine were obtained in the 150 enrolled study participants (see text for additional enrollment details). 50
The LC-MS/MS assay performance for measuring iohexol and iothalamate in plasma and urine was excellent, with coefficients of variation , 3% and Am J Kidney Dis. 2016;67(1):49-55
Iothalamate and Iohexol Urinary Clearances
recoveries generally .99% across all concentration ranges. Full details of the assay performance are provided in Item S1. Figure 1 summarizes the enrollment of study participants for concurrent measurement of urinary clearances of iothalamate, iohexol, and creatinine. Clinical characteristics of the study population are included in Table 1. More men (n 5 81) than women (n 5 69) were enrolled in the study. Mean age was 54 6 13 (range, 22-81) years. Forty percent (n 5 60) of study participants had previously undergone solid-organ or bone marrow transplantation. Indications for testing in the rest included potential kidney donor evaluation, pre–liver or pre–bone marrow transplantation evaluation, and chronic kidney disease (CKD) staging. Iohexol versus iothalamate clearances are compared in Fig 2A, whereas Figure 2B and C shows creatinine versus iothalamate and iohexol clearances, respectively, for all participants enrolled in the study. Passing-Bablok regression was used to assess these relationships because it is a robust form of regression that places less weight on outlier observations. Of note, the association between iohexol and iothalamate clearances had significant proportional bias (slope 5 0.85; 95% CI, 0.83-0.88), but not a significant constant bias (intercept 5 0.44; 95% CI, 0.43-1.50; Fig 2A). The proportional bias for iothalamate clearances compared with creatinine clearances was significantly less than for iohexol, although both were statistically Table 1. Study Population Characteristics Parameter
No. of participants Age, y Sex, M:F Race White African American Other
Value
150 54 6 13 (22-81) 81:69 91% 1% 8%
Diagnoses Diabetes mellitus Kidney transplant recipient Other transplant recipient Primary glomerular disease Polycystic kidney disease Pre– or post–kidney donor Amyloidosis/multiple myeloma Other
16 (11) 39 (26) 34 (23) 7 (5) 5 (3) 11 (7) 8 (5) 30 (20)
Clearance, mL/min/1.73 m2 Iothalamate Iohexol Creatinine
60 6 34 52 6 29 74 6 40
Note: Unless otherwise indicated, values for categorical variables are given as number (percentage); for continuous variables, as mean 6 standard deviation or mean 6 standard deviation (range). Am J Kidney Dis. 2016;67(1):49-55
significant (slopes of 1.09 [95% CI, 1.03-1.15] and 1.27 [95% CI, 1.20-1.34], respectively). In addition, the constant biases for both iothalamate and iohexol clearances compared with creatinine clearances were both statistically significant (intercepts of 5.27 [95% CI, 2.66-8.60] and 4.63 [95% CI, 1.78-7.68], respectively), resulting in both iohexol and iothalamate clearances being less than creatinine clearance, as expected (Figs 2B and C). Iothalamate and iohexol concentrations were comparable to each other in both plasma and urine (Table 2). By Bland-Altman plot, iothalamate clearance displayed an average 15% bias (P , 0.001) compared to iohexol (Fig 3A). This consistent bias was observed throughout the range of GFRs evaluated. The average bias of creatinine versus iothalamate was 24% (Fig 3B), whereas for creatinine versus iohexol, it was 38% (Fig 3C). To further investigate the consistent 15% difference between renal clearance of the 2 agents, iothalamate and iohexol were added to 10 clinical laboratory plasma waste samples and dialyzed against PBS. The fraction of iohexol in the plasma after versus before overnight dialysis was significantly higher than for iothalamate (0.19 6 0.03 vs 0.11 6 0.02, respectively; P , 0.001; Table 3). These data suggest that iohexol is not as ultrafilterable across a 2,000-Da membrane as iothalamate, perhaps due to differences in protein binding or other as yet undefined differences in diffusibility. Although the ratio of iohexol to iothalamate urinary clearance was not associated with sex (P 5 0.8), it was statistically significantly associated with age (P 5 0.03), but not necessarily clinically associated with age, as evidenced by the very small coefficient of determination (R2 5 0.03).
DISCUSSION The most notable finding of this study is the unequivocal demonstration that urinary clearance of iohexol is on average 15% less than that of iothalamate across a wide range of GFRs. Furthermore, iohexol is less ultrafiltrable than iothalamate across a 2,000-Da molecular weight dialysis membrane in vitro, which likely contributes to their differential clearance by the kidney in vivo. These findings have important implications for the comparison of renal clearances obtained using these 2 methods or when pooling them for larger meta-analyses. In the current study, we developed a new LC-MS/ MS assay to simultaneously analyze iothalamate and iohexol in urine and plasma specimens. This complements our previous work on the development and validation of a similar LC-MS/MS assay for iothalamate alone.15 In developing the LC-MS/MS assay for concurrently measuring iothalamate and iohexol in urine and plasma, minor methodologic changes were 51
Seegmiller et al
Figure 2. Passing-Bablok regression comparison plots of concurrent urinary clearances of (A) iohexol versus iothalamate, (B) creatinine versus iothalamate, and (C) creatinine versus iohexol. The equations for the regression lines (with 95% confidence intervals (CIs) of slope and intercept, respectively, in parentheses) are (A) y 5 0.85x 1 0.44 (0.83 to 0.88; 20.43 to 1.50); (B) y 5 1.09x 1 5.27 (1.03 to 1.15; 2.66 to 8.60); and (C) y 5 1.27x 1 4.63 (1.20 to 1.34; 1.78 to 7.68). Solid black lines are Passing-Bablok regression; dashed lines are 95% CIs of the regression, and thin gray lines are lines of identity, x 5 y. Abbreviations: CreatClr, creatinine urinary clearance; IohexClr, iohexol urinary clearance; IothClr, iothalamate urinary clearance. 52
implemented to the original iothalamate analytical method that slightly improved the performance of the previously reported iothalamate assay; the end result is excellent analytical sensitivity and specificity. Previously our laboratory and others have used capillary electrophoresis to measure iothalamate and/ or iohexol. However, the major advantage of LC-MS/ MS over capillary electrophoresis is greater analytic specificity and hence less susceptibility to interfering substances. A major objective of the current study was to better delineate possible differences between the renal clearances of iothalamate and iohexol. Few studies have directly compared the renal clearances of iothalamate and iohexol. Moreover, typically iohexol clearances are measured by way of plasma clearance, whereas iothalamate is more commonly used with urinary clearance methods. Only one other study directly compared simultaneous renal clearance of iothalamate and iohexol.5 In this small study performed with 14 patients and 3 healthy volunteers, Isaka et al5 reported that the slope of iothalamate versus inulin clearance was 1.06 6 0.02, whereas that for iohexol versus inulin clearance was 0.83 6 0.02. Soveri et al16 recently reviewed prior published reports of the accuracy of GFR measurement methods using renal inulin clearance as reference. Although none of the reported studies simultaneously compared iothalamate, iohexol, and inulin, they noted a mean bias for urinary clearance of iohexol versus inulin of 218 at GFR of 30, 211 at GFR of 60, and 22 at GFR of 90 mL/min/1.73 m2, with slightly positive mean bias of iothalamate across this range of GFRs (17, 16, and 15 mL/min/1.73 m2, respectively). The reported overall mean bias was 211 for iohexol and 16 for iothalamate, a difference of 17 mL/min/ 1.73 m2 between the 2 and thus not too dissimilar to the results of the current study. Interestingly, there were more studies for renal clearance of iothalamate (n 5 13) than iohexol (n 5 2), whereas the reverse was true for plasma disappearance (n 5 1 vs n 5 5, respectively). The authors also cautioned that many of the studies were small. However, the pooled results support the direction of the bias between the 2 methods that we observed here, with iohexol urinary clearance being less than iothalamate urinary clearance. The explanation for the observed difference between iothalamate and iohexol urinary clearance is not certain. Ideally, inclusion of inulin clearance as the goldstandard clearance marker would have been helpful. However, at the time of this study, inulin was not available as an approved injectable substance for humans. Thus, we cannot be certain whether urinary clearance of iothalamate or iohexol is a more accurate representation of GFR. It is interesting to note that early works by Maher et al3 and Sigman et al1 demonstrated Am J Kidney Dis. 2016;67(1):49-55
Iothalamate and Iohexol Urinary Clearances Table 2. Ratios of Urine-Plasma Concentrations for Iothalamate, Iohexol, and Creatinine Marker
Mean 6 SD
Iothalamate Iohexol Creatinine
1.31 6 1.13 1.12 6 0.93 1.76 6 1.71
Abbreviation: SD, standard deviation.
that urinary clearance of inulin and radiolabeled iothalamate were nearly identical across a wide range of GFRs, as did the more recent study of Isaka et al.5 The current study included creatinine clearance as a potential comparator. The bias between iothalamate and creatinine clearance in the current study (24%) is exactly that observed between inulin and creatinine clearance in the study by Isaka et al,5 whereas in the current study, the bias between iohexol and creatinine clearance is much greater (creatinine clearance 38% greater than iohexol). These observations suggest that iothalamate clearance may be closer to measured inulin clearance than iohexol clearance. The current study also suggests that decreased ultrafilterability of iohexol compared to iothalamate, perhaps due to increased plasma protein binding, could account for its relatively lower clearance, a possibility that has been previously discounted by others.8,17 Of note, clearances for both markers have been observed to be higher when measured by plasma disappearance as compared with urinary clearance.8 This also has been the case for inulin18,19 and has previously been attributed to ongoing extrarenal clearance of the markers in the periphery. Future studies comparing renal clearances of iothalamate, iohexol, and inulin, when possible, would be enlightening. A difference in the absorption rate of the 2 analytes from the subcutaneous depot could also potentially result in a difference in clearance results of the 2 analytes. Because the mean concentration from a pooled pre- and postclearance plasma sample was used as the plasma concentration for each clearance measurement, we cannot be certain that the mean value is the most accurate representation of the plasma concentration during the urine collection period for both analytes. Although not a focus of this study, the comparison of creatinine clearances with both iothalamate and iohexol clearances showed, as expected, that creatinine clearances were greater than those of both of the other 2 markers. This is in keeping with multiple other reports, including the recent analysis by Soveri et al.16 Because only relatively recently have standardized creatinine assays been implemented, the older reports may affect direct comparability with the current study. In addition to the lack of availability of the reference marker inulin to assess accuracy, use of a single urine collection of approximately one hour duration for the Am J Kidney Dis. 2016;67(1):49-55
Figure 3. Relative difference (Bland-Altman) plots of concurrent clearances of (A) iohexol versus iothalamate, (B) creatinine versus iothalamate, and (C) creatinine versus iohexol. The x-axis is the average of the 2 respective clearances, the y-axis is the difference of the 2 clearance measurements divided by the average (adjusts for increasing dispersion of measurements with increasing glomerular filtration rate). Solid black lines represent the mean relative difference (%); dashed lines, the 95% confidence limits of agreement; and thin gray lines, present bias 5 0. Abbreviations: CreatClr, creatinine urinary clearance; IohexClr, iohexol urinary clearance; IothClr, iothalamate urinary clearance.
53
Seegmiller et al Table 3. Fraction of Iothalamate and Iohexol Remaining in Plasma Following Overnight In Vitro Dialysis Experiments Marker
Mean 6 SD
Pa
Iothalamate Iohexol
0.11 6 0.02 0.19 6 0.03
,0.001
Abbreviation: SD, standard deviation. By paired t test.
a
clearance measurements is a potential weakness. As such, precise GFR measurement would be strengthened with longer or multiple urine collection periods. However, the goal of the current study was not accurate determination of GFR per se, but rather comparison between the 2 commonly used index GFR methods (iothalamate and iohexol). Simultaneous measurement of both iothalamate and iohexol in the same urine and plasma aliquots controlled for most preanalytic issues such as timing of collections, bladder emptying, and sample processing and thus minimized the influence of those variables in the comparison of the 2 analytes. Regardless of their relative bias compared to inulin clearance, the current study has obvious relevance for comparing data in clinical studies in which GFR was assessed by renal clearance of different markers. Because of the difficulty performing renal clearances from large cohorts, pre-existing clinical data are often pooled, often including various GFR methodologies. Consideration must be given to the potential implications of using pooled data across differing GFR measurement methodologies. For example, to develop the recent CKD-EPI (CKD Epidemiology Collaboration) equations, GFR was measured using iothalamate in 82%, iohexol in 9%, and EDTA in 9%.20 However, most studies using iohexol for GFR measurement rely on plasma clearance measurements rather than urinary clearance, the latter of which was the comparison in the present study. Soveri et al16 noted a mean bias of 27 for plasma clearance versus 12 mL/min/1.73 m2 for urinary clearance of iohexol in relation to inulin clearance, although the number of patients studied in the latter group was limited. Thus, whether plasma versus urinary clearance of iohexol exhibits similar bias relative to iothalamate is unclear and worthy of further evaluation. In conclusion, we show that iohexol renal clearances are lower than iothalamate renal clearances. Decreased ultrafilterability of iohexol, perhaps due to greater protein binding, might contribute to the lower measurements.
ACKNOWLEDGEMENTS Support: None. Financial Disclosure: The authors declare that they have no relevant financial interests. Contributions: Research idea and study design: JCS, TSL; data acquisition: JCS, BEB, CAS; data analysis/interpretation: JCS, 54
JCL, TSL; supervision: TSL. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. TSL takes responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.
SUPPLEMENTARY MATERIAL Figure S1: Representative chromatogram of an iothalamate/ iohexol calibrator. Item S1: Supplemental tandem mass spectrometry methodology for measurement of iohexol. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2015.08.020) is available at www. ajkd.org
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