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Published: March 22, 2010
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Rapid detection of non-radioactive GFR markers
Determining glomerular filtration rate (GFR) is costly, time-consuming, and difficult to perform. A simple, rapid test has been developed to measure GFR markers accurately in blood and urine.
Chronic kidney disease (CKD) is a major medical problem in the United States and the rest of the world. In 2008, a 30% increase in CKD cases during the past decade prompted the U.S. Renal Data System (USRDS) to issue for the first time a separate report documenting the magnitude of the disease, which affects an estimated 27 million (or one in ten) Americans and accounts for more than 24% of Medicare costs.1 An earlier report also estimated that at least 20 million more people are at risk for CKD.2 According to the USRDS’ “2008 Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease,” the total cost incurred for end-stage kidney disease was $33.6 billion. This amount included Medicare spending and all expenditures by other payers, such as employer group health plans. In 2006, costs for Medicare patients with CKD exceeded $49 billion, nearly five times greater than costs in 1993.
Challenges of Measuring Kidney Function
Kidney disease increases the risk of heart and blood vessel disease, and the major causes of kidney dysfunction include hypertension, diabetes, lupus erythematosus, chemotherapy, and immunosuppression therapy.3 In all of these instances, having an accurate test for kidney function is essential to determine the most appropriate therapeutic intervention. Preventing or slowing the progression of renal disease through early recognition of impaired renal function can reduce the number of patients with end-stage renal disease. At the same time, such early recognition can prevent or reduce the need for costly procedures such as dialysis and kidney transplants.4
Glomerular filtration rate (GFR) is the best measurement of the efficiency of kidneys in removing waste and excess fluid from the blood. GFR is determined by measuring the disappearance of certain agents from the blood and their appearance in urine. Currently existing methods for measuring GFR are expensive and tedious, or require radioactive markers and specialized facilities. GFR is estimated from the urinary clearance of an ideal filtration marker, which is defined by Ci = (Ui × V)/Pi. In this equation, Ci is the clearance of the ideal filtration marker i, Ui is the urinary concentration of i, V is the urine flow rate, and Pi is the average plasma concentration of i during the time interval for the urine collection. If substance i is freely filtered across the capillary wall and is neither secreted nor reabsorbed, then Ci = GFR.
Filtration Markers
Endogenous Filtration Markers. The clearance of endogenous filtration markers (e.g., creatinine, cystatin C, and urea) has been used to assess GFR.5,6 Serum creatinine determination has become a mainstay in the standard laboratory profile for renal function because of its convenience and low cost. However, serum creatinine is a crude marker for GFR because creatinine concentrations are insensitive to detecting mild to moderate reductions in GFR, which is due to the nonlinear relationship between concentrations of creatinine in blood and GFR.7
Using serum creatinine levels as an index for GFR depends on the following three important assumptions: creatinine is an ideal filtration marker whose clearance approximates GFR, the creatinine excretion rate is constant among all persons and over time, and the measurement of serum creatinine is accurate and reproducible across all clinical laboratories. Although serum creatinine and cystatin C concentrations can provide a rough index for the level of GFR, none of these assumptions is absolutely correct. Numerous factors such as kidney disease, reduced muscle mass, ingestion of cooked meat, malnutrition, etc., can lead to errors in estimating the level of GFR from the serum creatinine concentration.8
Moreover, the method of using serum creatinine to assess kidney function, along with another biomarker, cystatin C, fails to measure accurately kidney function under several clinical situations, including the following: extremes in age and body size, severe malnutrition and obesity, skeletal muscle disease, paraplegia or quadriplegia, vegetarian diets, rapidly changing kidney function, and drugs with significant toxicity that are excreted by kidneys.9,10 In addition, several substances such as glucose, uric acid, ketones, plasma proteins, and cephalosporin may lead to falsely high creatinine values when the Jaffe colorimetric method is used.7,11 Accurate assessment of renal function is important for not only early detection of CKD but also many other situations, including chemotherapy and organ transplant.
Artificial GFR Markers. Inulin and iothalamate I-125 are considered the gold standards for determining GFR in clinical settings.4 However, inulin is expensive, and the protocols for measuring inulin clearance are complicated. The clearance of various radioisotope markers, including 99mTc-labeled DTPA, 51Cr-labeled EDTA, and 125I-labeled iothalamate, can also be used to assess GFR reliably. However, such methods are costly, and require specialized facilities and trained personnel to handle radioactive materials.7 While other methods for determining GFR include using non-radioactive iodinated X-ray contrast agents (e.g., iothalamate and iohexol), they require analytical methods such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and X-ray fluorescence (XRF).12-15
While the most commonly used procedures for assessing GFR involve HPLC and CE, these separation techniques are expensive and only suitable in laboratory settings since both require sophisticated instrumentation, regular maintenance, and highly skilled personnel. The total cost for measuring iothalamate clearance is about $200 using the HPLC method, which is considerably lower than the cost for Tc-99m-DTPA clearance, which is approximately $400. This significant cost difference is due to the stricter testing routines that have to be adopted when isotopes are involved. Thus, to assess precisely kidney function, a direct method is needed to measure GFR conveniently and accurately using a suitable non-radioactive marker.
Rapid Detection of Non-Radioactive Iodinated GFR
A simple device that rapidly and accurately measures non-radioactive, iodinated GFR markers (e.g., iothalamate in blood and urine) is being developed at Lynntech Inc. (College Station, TX). This device is based on an innovative method that combines electrochemistry, solid phase extraction, and colorimetry, which will enable clinicians to measure GFR conveniently. The chemistry behind this innovative approach is shown in Figure 1.
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| Figure 1. Three steps for the detection of iodinated GFR markers. |
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| Figure 2. Iodine captured as a yellow iodine-PVP complex on the PVP-coated membrane from samples containing 0, 10, 20, 50, and 100 ppm of iothalamate (top) and their reflectance absorbance spectra (bottom). |
Iodine-based GFR markers are electrochemically dissociated at a bismuth electrode in order to release iodine as a free iodide. The iodide is chemically oxidized to iodine, then filtered and passed through a polyvinylpyrrolidone (PVP)–coated membrane. Iodine adsorbs onto the PVP-coated membrane, forming a yellow iodine-PVP complex. The iodine-PVP complex is stable and has a much higher extinction coefficient compared to molecular iodine.16 Figure 2 shows a photograph of PVP-coated membranes and a set of reflectance absorbance spectra taken after extraction of the iodine from samples containing various concentrations of iothalamate. Quantification of the iodine-PVP complex is achieved by measuring the reflectance absorbance with an optical fiber probe and a spectrophotometer. A comparison of the features of this electro-colorimetric method for the rapid detection of GFR markers with other existing methods is presented in Table I.
Selecting Electrode Material. In order to release iodide, electrochemical reduction of aryl iodide can be achieved by careful selection of the electrode material and special design of the electrochemical cell. For this electrochemical reduction, mercury or bismuth electrodes can be used because of their high over-potential for hydrogen reduction. Such high over-potential allows for the application of sufficient negative potential in an aqueous medium to reduce aryl iodide with negligible breakdown of the solvent. Although mercury has a higher over-potential for hydrogen reduction, a bismuth electrode was chosen for the device because of the toxicity and disposal problems associated with mercury and mercury compounds.
Electrochemical Cell Design. In a regular electrochemical cell with a single compartment, iodide that is produced by the electrochemical reduction of aryl iodide at the bismuth working electrode can be oxidized to iodine at the platinum counter electrode and escape as gaseous iodine. To prevent this from happening, a two-compartment cell separated by a Nafion membrane was constructed for the device. The Nafion membrane separating the two compartments allows charge transfer (proton exchange), but because of its negatively charged surface, it repels all anionic species. Thus, all anions including iodide cannot pass through the membrane and will be retained in the compartment.
Chemical Oxidation. An aliquot (200 μL) of the electrolyzed sample is forced through a glass microfiber filter that is preloaded with a powdered oxidant, or oxone. The iodide that is released from the GFR marker is chemically oxidized to iodine in the filter.
Iodine-PVP Complex Formation. The iodine-containing solution is immediately passed through a PVP-coated membrane, where molecular iodine is quantitatively extracted as an iodine-PVP complex on the top surface, thereby facilitating highly sensitive colorimetric detection of iodine. The resulting iodine-PVP complex is less volatile and more intensely colored than molecular iodine.
An additional advantage of this approach is that the measurement of the iodine-based GFR markers is not affected by other sources of iodide or iodine, such as iodinated salt ingested by patients or yellow-colored substances that may be present in urine. Although free iodide or iodine present in a sample can be measured prior to the electrochemical dissociation and subtracted from the total iodine measured, because of the low iodide concentrations in plasma and urine (less than 1 ppm in most cases), this correction is not required.17,18
Experiments
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| Figure 3. Lynntech’s GFR marker detection system using disposable components. |
The two-compartment electrochemical cell and disposable cartridges in the device developed for chemical oxidation and colorimetric determination of the iodinated GFR markers are shown in Figure 3.
Lynntech’s optimized process for measuring iothalamate is outlined in Figure 4.
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| Figure 4. A flow diagram showing the workflow of GFR marker detection in plasma and urine samples. |
The block diagram shows two parallel processes in which plasma and urine analysis requirements are slightly different. When plasma or bovine serum albumin (BSA) is mixed with perchloric acid, a precipitate is formed which must be removed by centrifugation, while urine analysis requires 4–8 times more oxone than plasma or BSA. Otherwise, the processes are identical.
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| Figure 5. The absorbance values for iothalamate in BSA are close to half of that for iodide, which corresponds to quantitative recovery of iodine from iothalamate. |
The results from tests performed with various concentrations of iodide and iothalamate in 4% BSA are shown in Figure 5. Measured at a wavelength of 390 nm, the absorbance values for the iothalamate samples were about half of those values for the iodide samples at the same concentration. Since approximately half (47%) of the molecular mass of iothalamate meglumine is iodine, this result indicates that iodide is released quantitatively from iothalamate by the electrochemical process.
Detecting an Iodinated GFR Marker in Plasma
Concentrations of iothalamate that spiked in human plasma samples were successfully detected using optimized parameters for measurements in 4% BSA (see Figure 4). 500 μL each of plasma sample and 4% perchloric acid were added to a centrifuge tube, vortexed to mix, and centrifuged at 15,000 rpm for one minute to separate the precipitated proteins. An 800-μL aliquot of the clear supernatant solution was pipetted into the sample compartment of the two-compartment electrochemical cell. A platinum coil electrode was placed into the other compartment, which was separated by the Nafion membrane, and filled with a 1:1 mixture of 4% perchloric acid and pH 7.4 PBS buffer.
The bismuth electrode was cleaned and placed into the sample compartment, and the sample solution was stirred with a magnetic stirrer. A regulated power supply applied 3.6 V to the bismuth electrode for five minutes, after which a 200-μL aliquot of the electrolyzed solution was passed first through the glass microfiber filter loaded with 0.5 mg of oxone and then through the PVP-coated membrane. The amount of iothalamate present in the sample was measured from the absorbance by the iodine-PVP complex. For human plasma samples spiked with 0, 10, 20, 50, and 100 ppm of iothalamate, baseline-corrected absorbance at 390 nm is shown in Figure 6. The linear fit for this concentration range showed a correlation coefficient of 0.981 with an intercept of 0.026 ppm.
Detecting an Iodinated GFR Marker in Urine
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| Figure 6. Standard curve for the detection of iothalamate in human plasma. The values shown are the average of three runs, and the plot is the linear fit of the data in the range of 0-100 ppm iothalamate. |
Due to negligible protein concentration in urine compared with plasma, urine samples do not require centrifugation. However, urine samples that spiked with iothalamate failed to develop any color when tested under the conditions optimized for plasma samples. This result arose from the oxidizer (0.5 mg of oxone loaded in the glass microfiber filter) being completely consumed by urea present in the urine sample, which caused incomplete oxidation of iodide released from iothalamate. The iothalamate concentration in the urine samples could be successfully measured by quadrupling the amount of oxidant loaded on the glass filter.
Average values for the baseline-corrected absorbance at 390 nm that were obtained from three sets of urine samples spiked with 0-, 10-, 20-, 50-, and 100-ppm iothalamate are plotted in Figure 7. Although this plot is non-linear at the current developmental stage, a high degree of reproducibility (small error bars) was obtained. Therefore, this plot may be used for accurate detection of iothalamate in urine samples within the concentration range studied.
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| Figure 7. Standard curve for the detection of iothalamate in human urine. |
Conclusion
The growing economic and societal burden caused by CKD demands a test method for rapidly detecting GFR markers in near-patient situations. A simple, inexpensive device that uses disposable cartridges, Lynntech’s electro-colorimetric technology makes it possible to quantify accurately iodinated GFR markers in both plasma and urine samples in clinical settings. With this method, iothalamate in plasma and urine can be determined rapidly and accurately in less than ten minutes.
References
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17. H Namba, et al., “Evidence of Thyroid Volume Increase in Normal Subjects Receiving Excess Iodide,” Journal of Clinical Endocrinology and Metabolism 76, no. 3 (1993): 605-608.
18. D Nacapricha, et al., “Kinetic Determination of Iodine in Urine Using Stopped-Flow Injection,” Analytical Sciences 17 (2001): i33-i36.
Bikas Vaidya, PhD, is a senior research scientist at Lynntech Inc. (College Station, TX). He can be reached at bikas.vaidya@lynntech.com.
James Magnuson is a research associate at Lynntech Inc. (College Station, TX). He can be reached at james.magnuson@lynntech.com.
Season Wong, PhD, is a senior research scientist at Lynntech Inc. (College Station, TX). He can be reached at season.wong@lynntech.com.Rajiv Agarwal, MD, is a professor of medicine at Indiana University School of Medicine (Indianapolis, IN). He can be reached at ragarwal@iupui.edu.
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