Estimation of glomerular filtration rate in dogs by plasma clearance of gadolinium diethylenetriamine pentaacetic acid as measured by use of an ELISA.

OBJECTIVE-To evaluate use of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) to estimate glomerular filtration rate (GFR) by plasma clearance and use of an ELISA as the method of Gd-DTPA quantification. ANIMALS-16 dogs of various sexes and breeds (12 dogs were clinically normal, and 4 dogs were polyuric and polydipsic with no other clinical or biochemical abnormalities). PROCEDURES-GFR was estimated by measuring the plasma clearance of Gd-DTPA and iohexol by use of an ELISA and high-performance liquid chromatography (HPLC), respectively. The GFR was determined by use of a 1-compartment model for both methods. The GFRs obtained by Gd-DTPA plasma clearance were compared with those obtained by iohexol plasma clearance by use of correlation analysis, paired t tests, and limits of agreement analysis. A paired t test was used to evaluate differences between the 2 plasma clearance methods. RESULTS-A strong linear correlation (r(2) = 0.90) was found between GFRs derived from the plasma clearance of Gd-DTPA and those derived from the plasma clearance of iohexol. By use of limits of agreement analysis, almost all (13/14) dogs had Gd-DTPA GFRs that were within 12% of iohexol GFRs. The remaining dog had a Gd-DTPA GFR that was 45% higher than the iohexol GFR. There was no significant difference between Gd-DTPA GFRs and those obtained with iohexol. CONCLUSIONS AND CLINICAL RELEVANCE-This study revealed that plasma clearance of Gd-DTPA measured by use of an ELISA is an effective method to estimate GFR in dogs because it compared favorably with results for the iohexol-HPLC method.

Polycationic nanoparticles: (1) synthesis of a polylysine-MION conjugate and its application in labeling fibroblasts.

Nanoparticles are increasingly used to label cells to track them by imaging or to quantify them in vivo. However, normal cellular uptake mechanisms are inadequate to load cells with tracking label. We propose a simple method to coat nanoparticles, such as monocrystalline iron oxide nanoparticle (MION), with the transfection agent polylysine in order to facilitate rapid, uniform, and heavy labeling of fibroblasts. The method is based on commercially available reagents, requires no more than 1 h of laboratory contact time, and can be accomplished safely without a chemical hood. A suspension of MION was treated by addition of solid sodium periodate to oxidize glucose residues of dextran and introduced aldehyde groups to the dextran coat surrounding MION's crystalline magnetite core. After a 30-min incubation to effect oxidation, unreacted periodate was quenched with glycerol. The preparation was dialyzed to remove reactants and diluted to a final concentration of 2 mg Fe/ml. Poly-L-lysine was added to the oxidized MION (MION-A) to form reversible covalent Schiff base linkages. The resulting conjugate, a polylysine iron oxide nano-particle is abbreviated PLION. NIH3T3 fibroblasts labeled with either MION, MION-A, or MION plus polylysine showed minimal uptake of iron while cells labeled with PLION acquired a brown hue demonstrating strong labeling with iron. Microscopic assessment of iron labeling was confirmed using Prussian blue staining. In some cells, the concentration of iron was sufficiently high and localized to suggest association with cytoplasmic vacuoles. The nucleus of the cell was not labeled. Cell labeling increased when the ratio of polylysine to MION increased and with increasing amount of PLION.

Cell tracking using nanoparticles.

Tracking cells in regenerative medicine is becoming increasingly important for basic cell therapy science, for cell delivery optimization and for accurate biodistribution studies. This report describes nanoparticles that utilize stable-isotope metal labels for multiple detection technologies in preclinical studies. Cells labeled with nanoparticles can be imaged by electron microscopy, fluorescence, and magnetic resonance. The nanoparticle-labeled cells can be quantified by neutron activation, thereby allowing, with the use of standard curves, the determination of the number of labeled cells in tissue samples from in vivo sources. This report describes the characteristics of these nanoparticles and methods for using these nanoparticles to label and track cells.

Validation of neutron activation as a novel method to determine glomerular filtration rate.

BACKGROUND/AIMS: Despite widespread interest in determining the glomerular filtration rate (GFR) of patients, current methods all have significant limitations. Therefore, a compelling need exists for new tests of GFR that are both accurate and easy to perform. We have previously reported that the technique of neutron activation (NA) accurately measures iohexol in vitro. In this study, we demonstrate that NA can be used to determine GFR by measuring the clearance of iohexol, and directly compare these results to a gold-standard method based on (99m)Tc-DTPA. METHODS: We studied 57 patients with mild to moderate chronic kidney disease and normal volunteers. Subjects were simultaneously injected with iohexol and (99m)Tc-DTPA. Blood and urine samples were collected over 4 h to calculate GFR by the UV/P method. RESULTS: The range of GFRs was 28-212 ml/min. GFRs obtained using iohexol and (99m)Tc-DTPA correlated closely (R = 0.95). The bias between the 2 techniques was 0.96 ml/min, and precision (defined as the standard deviation of the mean of the difference between the 2 values for each patient) was 10.6 ml/min. Accuracy was such that 98% of subjects had NA GFRs within 20% of the reference (99m)Tc-DTPA measurements. CONCLUSIONS: We conclude that NA is an excellent technique to measure GFR. NA has several advantages over current methods to directly measure GFR, including the ability to reassay samples, high throughput and the avoidance of patient and hospital radioactivity exposure. In the future, NA could be applied to GFR agents that do not contain iodine, such as Gd-DTPA, and to the simultaneous measurement of agents that reflect renal blood flow, such as iodohippurate. Therefore, NA holds great potential to improve the measurement of renal function in a safe, easily obtainable way.

Ultrasmall mixed ferrite colloids as multidimensional magnetic resonance imaging, cell labeling, and cell sorting agents.

One area that has been overlooked in the evolution of magnetic nanoparticle technology is the possibility of introducing informational atoms into the iron oxide core of the coated colloid. Introduction of suitable atoms into the iron oxide core offers an opportunity to produce a quantifiable probe, thereby adding one or more dimensions to the magnetic colloid's informational status. Lanthanide-doped iron oxide nanoparticles have been synthesized to introduce informational atoms through the formation of colloidal mixed ferrites. These colloids are designated ultrasmall mixed ferrite iron oxides (USMIOs). USMIOs containing 5 mol % europium exhibit superparamagnetic behavior with an induced magnetization of 56 emu/g Fe at 1.5 T, a powder X-ray diffraction pattern congruent with magnetite, and R1 and R2 relaxivity values of 15.4 (mM s) (-1) and 33.9 (mM s) (-1), respectively, in aqueous solution at 37 degrees C and 0.47 T. USMIO can be detected by five physical methods, combining the magnetic resonance imaging (MRI) qualities of iron with the sensitive and quantitative detection of lanthanide metals by neutron activation analysis (NA), time-resolved fluorescence (TRF), X-ray fluorescence, along with detection by electron microscopy (EM). In addition to quantitative detection using neutron activation analysis, the presence of lanthanides in the iron oxide matrix confers attractive optical properties for long-term multilabeling studies with europium and terbium. These USMIOs offer high photostability, a narrow emission band, and a broad absorption band combining the high sensitivity of time-resolved fluorescence with the high spatial resolution of MRI. USMIO nanoparticles are prepared through modifications of traditional magnetite-based iron oxide colloid synthetic methods. A 5 mol % substitution of ferric iron with trivalent europium yielded a colloid with nearly identical magnetic, physical, and chemical characteristics to its magnetite colloid parent.

Method to quantify tail vein injection technique in small animals.

Injection errors, which are often not readily recognized, can greatly impact the outcome of a pre-clinical research study. As a result, unrecognized misadministration of test compounds can render a high cost to the biomedical community. In this report, we propose six criteria for a reagent designed to assess tail vein injection technique in small animals and suggest a reagent, colloidal gold labeled with the stable isotope 197Au, that satisfies these criteria, thereby describing and validating for the first time a method to quantify technical compliance in tail vein injections. In an application of this reagent, we show the degree of variation experienced by technologists performing tail vein injection procedures in mice. In this study, mice were manually restrained and received an injection in the tail vein. One hour after injection, the mice were euthanized, various organs including the tail (the site of the injection) were collected, and their gold content was quantified by neutron activation. The three experienced animal technologists in the study were tested for tail vein injection proficiency in 30 mice. Prior to the study, the supervisor stated that a misinjection occurs when more than 10% of the intended volume remains in the tail. In light of this criterion, 12 of the 30 injections were misadministered: two with technologist 1, three with technologist 2, and seven with technologist 3. Although she was able to correctly rank the injection skills of the three technologists used in this experiment, i.e., technologist 1 and 2 more better skilled than technologist 3, the supervisor greatly underestimated the extent and degree of injection failures for the procedure. The results of the study illustrate the potential problems associated with the technical compliance with this common laboratory procedure and suggest that there is a need to validate injection methods and a need to monitor technical competence. Application of reagents similar to colloidal gold and the methods presented will facilitate the development of improved methods of teaching injection technique and monitoring technical quality in the laboratory setting. In Vivo Micro Computed Tomography of Subchondral Bone in the Rat After Intra-articular Administration of Monosodium Iodoacetate

Neutron-activation analysis: a novel method for the assay of iohexol.

Accurate measurement of glomerular filtration rate (GFR) is of great importance in both research and clinical medicine. Available methods require radiation exposure or are technically difficult, thereby limiting their utility. Recent work has validated plasma clearance of nonradioactive contrast agents (iohexol and iothalamate) for the measurement of GFR. However, their clinical utility has been restricted by the difficulties associated with the detection of contrast agents in serum. In this investigation, we evaluate the sensitivity, accuracy and precision of neutron activation analysis (NAA) to measure serum iohexol at concentrations necessary for estimating GFR. We subjected aliquots of serum containing 0 to 6470 microg/mL iohexol to neutron activation by placing them in a neutron beam for 1 minute. The activation process resulted in the elevation of iohexol's naturally abundant iodine 127 to iodine 128. The spontaneous decay of (128)I to xenon 128 (proportional to the amount of total iodine in the sample) was calculated by means of spectrographic analysis. The correlation between the predetermined elemental mass of iodine in the sample and that measured on NAA was then determined. A similar analysis was performed to establish the intra- and interday accuracy and precision, with multiple measurements taken over a single day and over the course of a month. We noted excellent correlation between iodine measured on NAA and the known elemental mass (r(2) =.99). Measurements were highly accurate (mean accuracy 2.4% +/- 1.8%), with excellent intra- and interday reliability (mean coefficient of variation 4.1% +/- 1.6%). NAA is a feasible and reproducible method of detecting iohexol for the purpose of measuring GFR.