Discordance Between Iothalamate and Iohexol Urinary Clearances.

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±28 (SD), 60±34, and 74±40 mL/min/1.73 m(2), 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.

Determination of iohexol and iothalamate in serum and urine by capillary electrophoresis.

Estimation of glomerular filtration rate (eGFR) is essential to assess kidney function. Iodine-containing contrast agents detection by HPLC has been proposed as a safe alternative for inulin or radioactive compounds. However, HPLC is a time-consuming and labor-intensive method. The aim of this study was to develop an assay for iohexol and iothalamate using capillary electrophoresis. Iohexol and iothalamate were directly analyzed by CE in serum and urine, using photometric detection (246 nm). Serum peak height was proportional to iohexol and iothalamate concentrations. Detection limits for iohexol and iothalamate were 10 and 5 mg/L. Limits of quantification were 13.0 and 15.0 mg/L. Within-run CVs were 4.9 and 6.5%; between-run CVs 3.1-9.9% and 3.8-13.7%. A good correlation was observed between CE and HPLC: y = 1.1703x + 5.017 (iohexol) and y = 0.7807x + 11.01 (iothalamate; (y = concentration obtained by CE [mg/L], x = concentration obtained by HPLC [mg/L]). In addition, CE allowed to determine urinary iohexol concentration. Although the detection limit for CE was higher than for HPLC, CE can still be used for eGFR determination. Advantages of this high-throughput method are the absence of sample pretreatment and a minimal sample volume requirement.

Cystatin C enhances glomerular filtration rate estimating equations in kidney transplant recipients.

BACKGROUND: The glomerular filtration rate (GFR) estimating equation incorporating both cystatin C and creatinine perform better than those using creatinine or cystatin C alone in patients with reduced GFR. Whether this equation performs well in kidney transplant recipients cross-sectionally, and more importantly, over time has not been addressed. METHODS: We analyzed four GFR estimating equations in participants of the Angiotensin II Blockade for Chronic Allograft Nephropathy Trial (NCT 00067990): Chronic Kidney Disease Epidemiology Collaboration equations based on serum cystatin C and creatinine (eGFR (CKD-EPI-Creat+CysC)), cystatin C alone (eGFR (CKD-EPI-CysC)), creatinine alone (eGFR (CKD-EPI-Creat)) and the Modification of Diet in Renal Disease study equation (eGFR (MDRD)). Iothalamate GFR served as a standard (mGFR). RESULTS: mGFR, serum creatinine, and cystatin C shortly after transplant were 56.1 ± 17.0 ml/min/1.73 m(2), 1.2 ± 0.4 mg/dl, and 1.2 ± 0.3 mg/l respectively. eGFR (CKD-EPI-Creat+CysC) was most precise (R(2) = 0.50) but slightly more biased than eGFR (MDRD); 9.0 ± 12.7 versus 6.4 ± 15.8 ml/min/1.73 m(2), respectively. This improved precision was most evident in recipients with mGFR >60 ml/min/1.73 m(2). For relative accuracy, eGFR (MDRD) and eGFR (CKD-EPI-Creat+CysC) had the highest percentage of estimates falling within 30% of mGFR; 75.8 and 68.9%, respectively. Longitudinally, equations incorporating cystatin C most closely paralleled the change in mGFR. CONCLUSION: eGFR (CKD-EPI-Creat+CysC) is more precise and reflects GFR change over time reasonably well. eGFR (MDRD) had superior performance in recipients with mGFR between 30 and 60 ml/min/1.73 m(2).

Iothalamate quantification by tandem mass spectrometry to measure glomerular filtration rate.

BACKGROUND: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. METHODS: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8-487.0, 614.8-456.0, 614.8-361.1, and 614.8-177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. RESULTS: Mean recovery was 90%-110% in both urine and plasma matrices. Imprecision was

Rapid analysis of iodinated X-ray contrast media in secondary and tertiary treated wastewater by direct injection liquid chromatography-tandem mass spectrometry.

The iodinated X-ray contrast media are the most widely administered intravascular pharmaceuticals and are known to persist in the aquatic environment. A rapid method using direct injection liquid chromatography-tandem mass spectrometry (DI-LC-MS/MS) has been developed to measure eight ICM. These include iopamidol, iothalamic acid, diatrizoic acid, iohexol, iomeprol, iopromide, plus both ioxaglic acid and iodipamide, which have not previously reported in the literature. The LC-MS/MS fragmentation patterns obtained for each of the compounds are discussed and the fragments lost for each transition are identified. Matrix effects in post-RO water, MQ water, tap water and secondary effluent have also been investigated. The DI-LC-MS/MS method was validated on both secondary and tertiary treated wastewater, and applied to samples from an advanced activated sludge wastewater treatment plant (WWTP) and a water recycling facility using microfiltration (MF) and reverse osmosis (RO) in Perth, Western Australia. As well as providing information of the efficacy for RO to remove specific ICM, these results also represent the first values of ICM published in the literature for Australia.

A natriuretic principle derived from kidney tissue of volume-expanded rats;.

Homogenates of kidneys from hydropenic and volume-expanded rats were subjected to gel filtration with Sephadex G-25. A fraction of the eluate coincident with the fourth UV peak was injected into the aorta of rats with one kidney excluded. A fraction eluting before the albumin peak was utilized as a control. Significant natriuresis and diuresis were observed after infusion of the fraction obtained from volume-expanded kidneys but not after infusion of the fraction from hydropenic kidneys or the control fraction. The natriuresis occurred in in the absence of changes in mean blood pressure, hematocrit, plasma sodium and potassium, glomerular filtration rate, and potassium excretion. The response was apparent immediately after infusion and persisted for up to 150 min. These results verify the existence of a low molecular weight natriuretic substance which may be preferentially bound to the kidney after its volume-stimulated release into the circulation.

High performance liquid chromatographic measurement of iothalamate in human serum and urine for evaluation of glomerular filtration rate.

A simple and sensitive HPLC-UV assay was developed for the measurement of iothalamate (IOT) in human serum and urine. Chromatographic separation was achieved using an embedded-carbamate-group bonded RP18 column and mobile phase consisting of 50 mM monobasic sodium phosphate and methanol (90:10, v/v) without the addition of ion-pair reagents. The assay demonstrated a high analytical reliability within the IOT concentration range of 1-150 microg/ml in serum and 25-1500 microg/ml in urine. The relative standard deviations (RSDs) for intra- and inter-day analysis were less than 5.1% in all cases. This method has been used for the evaluation of glomerular filtration rate (GFR) in subjects participating in a phase I clinical trial of a novel antimalarial medicine. The average baseline GFR was 100.41+/-19.99 ml/min/1.73 m(2) in 119 healthy volunteers. The assay may also allow the simultaneous measurements of p-aminohippuric acid (PAH), N-acetyl PAH (aPAH), and IOT with some modification. PAH, IOT, aPAH, and beta-hydroxyethyl-theophylline internal standard peaks appeared approximately at 2.5, 3.7, 5.9, and 11.8 min, respectively, in an isocratic run.

A simple LC-MS method for the determination of iohexol and iothalamate in serum, using ioversol as an internal standard.

BACKGROUND: Chronic kidney disease (CKD) is diagnosed and explored through the determination of the glomerular filtration rate (GFR). Our goal was to develop a simple LC-MS method for the determination in serum of 2 popular GFR markers, contrast agents iohexol and iothalamate, for routine use and comparison studies between the two markers. A similar contrast agent, ioversol, was used as an internal standard and the method underwent a rigorous validation protocol based on ß-expectation tolerance intervals. METHODS: We adapted the HPLC-UV method from Cavalier et al. to our LC-MS system. Data treatment for the validation was performed using Multiquant 3.0 (Sciex, Framingham, MA, USA) and e.noval 3.0 software (Arlenda, Liège, Belgium). RESULTS: According to the validation results our method will give accurate and reliable results for concentrations ranging from 6.8 to 250µg/ml for iohexol and 6.15µg/ml to 250µg/ml for iothalamate. In our practice these intervals are sufficient to determine both compounds in most patient samples. Samples with higher detected concentrations can always be diluted into range. CONCLUSION: With its internal standard and extensive validation, our method is now ready for routine and clinical research use.

Analysis of iodinated X-ray contrast agents in water samples by ion chromatography and inductively-coupled plasma mass spectrometry.

In this paper, an analytical method for the determination of six iodinated X-ray contrast agents (amidotrizoic acid, iohexol, iomeprol, iopamidol, iopromide, and ioxitalamic acid), iodide, and iodate in water samples is presented. The method is based on a separation of the analytes by ion chromatography (IC) and a subsequent detection by inductively-coupled plasma mass spectrometry (ICP-MS). The method was optimised with respect to separation conditions (column type and eluent composition) and extensively validated. Without pre-concentration of the samples, limits of detection below 0.2 microg/l could be achieved whereby reproducibility was below 6% for all compounds under investigation.

Simple HPLC-UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis.

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C(18) column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 microg/ml for iohexol and iothalamate, 5 to 40 microg/ml for PAH and 2.5 to 40 microg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.

Ambulatory GFR measurement with cold iothalamate in adults with chronic kidney disease.

BACKGROUND: The purpose of this study is to develop a method that eliminates the use of radioactive agents, reduces the length of inpatient visits, and does not require urine collection for the measurement of glomerular filtration rate (GFR) in adults with chronic kidney diseases (CKD). METHODS: We measured simultaneous urinary and plasma clearance of iothalamate using high-performance liquid chromatography in 30 patients with CKD after a continuous subcutaneous infusion of iothalamate at a rate of 125 microL/h for a 24-hour period. To ascertain whether a steady state was achieved with a 24-hour infusion, in 20 additional patients, we infused iothalamate continuously for 48 hours and measured GFR after 24 and 48 hours. RESULTS: Plasma iothalamate levels and urinary excretion rates obtained after 24- and 48-hour infusions were similar. GFR estimated by plasma clearance (44.4 mL/min; 95% confidence interval [CI], 37.0 to 53.3) was similar to urinary clearance (41.6 mL/min; 95% CI, 34.7 to 50.0) and without bias (average difference, 7%; 95% CI, +18% to -38%; P = 0.866). There also was acceptable agreement between plasma and urinary clearance of iothalamate (coefficient of variation [CV], 19.6% between techniques). Reproducibility of day-to-day plasma continuous infusion clearance technique was less than 12%. There was good agreement (CV, 19.9%) between inulin and renal iothalamate clearances. CONCLUSION: These data show that ambulatory infusion of iothalamate can be used to measure GFR in adults with CKD without the need for urine collections with acceptable precision and without exposure to radioactivity.

Development, chemistry, and physical properties of iopamidol and its analogues.

A series of 5-hydroxyacylamino-2,4,6-triiodo-N,N'-hydroxy-alkyl-isophtalamides was prepared in order to study the structural requirements for high water solubility and low toxicity. Among the amides substituted by 2-hydroxyethyl; 2,3-dihydroxypropyl; 1,3-dihydroxypropyl-; and 1,1-dihydroxy-methyl-2-oxyethyl- groups, the highest water solubility was obtained with the 1,3-dihydroxypropyl- group. Neither optical resolution of the 2,3-dihydroxypropyl moiety nor amide formation with two different amines improved the water solubility. The optimal solubility was obtained with S-5-alpha-hydroxypropionylamino-2,4,6-triiodoisophtalic acid di-(1,3-dihydroxy-2-propylamide), iopamidol. Iopamidol forms anhydrous and monohydrate crystals, characterized by differential thermal analyses, x-ray diffraction patterns, and solubility patterns. A method for enzymatic assay of the optical purity of iopamidol with lactodehydrogenase is described as well as partition coefficient, ionization constant of the oxyacylamido-group, critical micelle formation, surface tension, and osmolarity.

Faster and easier radiochemical purity testing for [125I]sodium iothalamate.

A previous method for determination of the radiochemical purity (RCP) value of [125I]sodium iothalamate uses two paper strips and solvents (total developing time is approximately 2.5 h). To simplify and shorten the RCP testing procedure, our laboratory has developed a single-strip chromatography method that not only distributes free 125I and [125I]sodium iothalamate to different relative front (Rf) locations, but is also faster and easier to perform. RCP of [125I]sodium iothalamate was determined with the use of a 10-cm instant thin-layer chromatography strip impregnated with polysilicic acid gel (ITLC-SA) as the solid phase, and a mobile phase of 2-butanol:acetic acid:water (140:2.5:70, v/v). By using autoradiography and counting the strip segments in a gamma counter, our results indicated that free 125I migrated to Rf = 0.89-1.00 while the [125I]sodium iothalamate moved to Rf = 0.44-0.67. The total developing time for the single-strip ITLC-SA system was approximately 1 h.

Column-switching liquid chromatographic method for the simultaneous determination of iothalamic acid and creatinine in biological fluids.

A column-switching liquid chromatographic method for simultaneous determination of iothalamate and creatinine in human serum and urine was developed. Iothalamate and creatinine were separated on a weakly acidic ion-exchange column (C1) by ion-exclusion chromatography and iothalamate excluded from the column was purified by gel chromatography on a hydrophilic gel column (C2) and then by ion-exchange chromatography on a weakly basic ion-exchange column (C3). Creatinine that was eluted from C1 after iothalamate was transferred to a hydrophilic gel column (C4) and then to a strongly acidic ion-exchange column (C5). The mobile phase for C1-C4 was a pH 3.8 propionate buffer (propionic acid-NaOH = 0.35 + 0.035 mol/kg in water) and a pH 5.6 propionate buffer (propionic acid-NaOH = 0.04 + 0.035 mol/kg in water) was used for C5. Diluted serum and urine samples could be injected directly on to C1, as the matrix of C1 is hydrophilic and C1 is backflushed after the transfer of the creatinine fraction from C1 to C4. Iothalamate and creatinine in the eluates were determined by measuring their ultraviolet absorption at 245 and 234 nm, respectively. The precision (R.S.D.) of the chromatographic method was 1.6% (n = 7) and 0.36% (n = 6) for diluted serum and urine with iothalamate concentrations of 1.0 and 10.0 mumol/l, respectively, and 0.85% (n = 7) and 0.55% (n = 7) for diluted serum and urine with creatinine concentrations of 5.77 and 272 mumol/l, respectively.

Liquid chromatography for iothalamate in biological samples.

We have previously reported an iothalamate assay for the assessment of the glomerular filtration rate (GFR) that required a long column equilibration time and 22 min run time per sample. We now report a simpler assay that requires a run time of only 5.5 min and is more precise and accurate than the earlier technique. The mobile phase consisted of methanol-acetonitrile-50 mM sodium monobasic phosphate (10:5:85, v/v) at pH 4.4, pumped at a rate of 1.5 ml/min on a C(18) reversed-phase column. Samples of plasma and urine were deproteinized with 1 volume of 4% perchloric acid or 9 volumes of 2% perchloric acid, respectively. No internal standard was used. The diode array detection system collected absorbance at 240 nm and the peak height areas of iothalamate were determined. The iothalamate peak appeared at 3.5 min. Detector response was linear over the range tested (10-2000 microg/ml). Within-run precision was <3% for both plasma and urine and accuracy was 96-102%. Between-day precision for plasma and urine analyses were <7%. The recovery of iothalamate in urine and plasma were 102% and 91%, respectively. There was excellent thermal and pH stability of iothalamate. No interference was found with para-amino hippuric acid (PAH) or N-acetyl PAH, which can be simultaneously assayed, if desired.

Radiopaque liposomes: effect of formulation conditions on encapsulation efficiency.

Liposomes containing sodium ioxitalamate were prepared by sonication. Suitable amounts of purified soybean phosphatidylcholine and cholesterol were used at various molar ratios. Stearylamine or dicetylphosphate were added to this lipid composition when charged liposomes were required. After sonication and removal of unencapsulated solute, this manufacturing process yielded small multilamellar vesicles as confirmed by electron microscopy. These liposomes did not exhibit a narrow range of size distribution; the mean particle size varied from 135 to 145 nm. With respect to the efficiency of encapsulation, two parameters were distinguishable: the volume of encapsulated aqueous space per unit of lipid weight and the percentage of the contrast agent added that became encapsulated in the liposomes. Investigation of the preparative parameters revealed that increased molar ratios of cholesterol yielded higher aqueous volume and iodine contents in the liposomes, which were attributed to a reduction of the liposome permeability to the contrast agent. However, the inclusion of cholesterol into the bilayer liposomal membrane was limited, probably by solubility restrictions. Negatively and positively charged liposomes had higher rates of encapsulation than did neutral liposomes. This result was expected since efficient encapsulation of polar compounds requires formation of large aqueous spaces within the vesicles per mole of lipids. Increase of the lipid fractions at a constant, reduced the aqueous volume entrapped per millimole of lipid and, consequently, the iodine content in the liposomes. However, an increase in the initial sodium ioxitalamate concentration diminished the aqueous volume entrapped in the liposomes but increased the iodine content.

Geriatric renal function: estimating glomerular filtration in an ambulatory elderly population.

In elderly individuals, serum creatinine may remain normal as glomerular filtration rate (gfr) declines. Therefore, the estimation of glomerular filtration utilizing mathematical models incorporates age as an important variable. In order to adjust drug dosages and diagnose renal disease earlier in the elderly, a variety of such simplified estimates of gfr have been applied. Unfortunately, no estimator is as accurate as the cumbersome gold standards (e.g. inulin or iothalamate clearance) and the reliability of each may vary with the particular clinical setting. The purpose of this study was to critically evaluate three commonly used estimators of gfr-i.e., creatinine clearance (CC), Cockroft-Gault (CG), and 100 over serum creatinine (100/SC)-comparing them to iothalamate clearance (IC) in a group of healthy ambulatory geriatric subjects (n = 41; ages 65-85). IC declined 1 ml/min per year of age in our sample. CC demonstrated a similar decline, a correlation of 0.83 with IC, and moderate error relative to IC of 17% at the mean (standard error [SE] = 12.3). In contrast, 100/SC correlated only 0.56 with IC, demonstrated a large positive bias (41 ml/min), and showed no age-related decline. An age correction to 100/SC similar to that utilized in the CG formula was clearly necessary. Despite the age and weight correction used in the CG formula, we found the estimates from it to be inaccurate (correlation = 0.5; SE = 23.8). A simpler age-corrected formula (Est. IC = 1/2 [100/SC] + 88-age) was derived and proved significantly superior to CG in our ambulatory geriatric sample, but still exhibited enough error (SE = 16.4) to question its clinical utility. It appears that serum creatinine based estimates of gfr in the elderly may not provide accurate results.

Acute effects of increasing doses of nicorandil on renal function in man.

The effects of nicorandil, a nicotinamide derived vasodilator combining nitrate and potassium channel opener actions, on kidney function have not been determined. This study investigated changes in renal blood flow and glomerular filtration rate as estimated using simultaneous 131I-iodohippurate and 125I-iothalamate plasma clearances. Forty-two healthy subjects in sodium balance received placebo and 2.5 mg (n = 8), 5 mg (n = 9), 10 mg (n = 8), 20 mg (n = 8) or 30 mg (n = 9) nicorandil orally. Peak nicorandil plasma concentrations occurred in the first hour. Nicorandil produced dose related decreases in blood pressure with maximum reductions (mean +/- standard error of the mean) after 30 mg of -6 +/- 1 mmHg systolic and -8 +/- 2 mmHg diastolic. Renal blood flow averaged 655 +/- 28 ml/minute/1.73 m2 after placebo. Renal blood flow changed 10 +/- 11% after 2.5 mg, -6 +/- 8% after 5 mg, -12 +/- 11% after 10 mg, -11 +/- 5% after 20 mg, and 8 +/- 6% after 30 mg, however, these changes did not reach statistical significance. Glomerular filtration rate averaged 113 +/- 3 ml/minute/1.73 m2 and was unaltered after nicorandil. Nicorandil had no effect on filtration fraction but fractional excretion of sodium tended to decrease with dose. These dose-related effects of nicorandil are consistent with other mixed vasodilators. At therapeutic doses, renal perfusion and function are preserved despite reductions in systemic blood pressure by nicorandil.

Paired ion reversed-phase HPLC assay for the determination of iothalamic acid and para aminohippuric acid in urine.

A paired ion reversed-phase high performance liquid chromatographic method for simultaneous determination of iothalamic acid (Io) and para aminohippuric acid (PAH) in urine is described. The method uses a single internal standard for both drugs. The only sample preparation required is dilution of urine (1:100 or 1:500) with deionized water. The internal standard is added to a small aliquot of the diluted specimen and injected. For HPLC, a C8 column and a mobile phase consisting of potassium phosphate buffer with dodecyl triethylammonium phosphate IP reagent, 25% organic modifier with UV detection at 254 nm was used. Within day and between day variation for the assay were in the range of 1.48-9.46% for iothalamic acid and 1.84-10.36% for para aminohippuric acid for four levels of concentration. Limits of quantitation were 50.0 micrograms ml-1 for iothalamic acid and 75.0 micrograms ml-1 for para aminohippuric acid. Mean recovery was 98.55% for Io and 97.79% for PAH. This isocratic HPLC assay is simple, rapid and relatively inexpensive.