Ethyl-iophenoxic acid as a quantitative bait marker for small mammals.

Bait markers are indispensable for ecological research but in small mammals, most markers are invasive, expensive and do not enable quantitative analyses of consumption. Ethyl-iophenoxic acid (Et-IPA) is a non-toxic, quantitative bait marker, which has been used for studying bait uptake in several carnivores and ungulates. We developed a bait with Et-IPA, assessed its palatability to common voles (Microtus arvalis), and determined the dose-residue-relation for this important agricultural pest rodent species. Et-IPA concentrations of 40 to 1280 µg Et-IPA per g bait were applied to wheat using sunflower oil or polyethylene glycol 300 as potential carriers. In a laboratory study, common voles were offered the bait and blood samples were collected 1, 7, and 14 days after consumption. The samples were analyzed with LC-ESI-MS/MS for blood residues of Et-IPA. Sunflower-oil was the most suitable bait carrier. Et-IPA seemed to be palatable to common voles at all test concentrations. Dose-dependent residues could be detected in blood samples in a dose-dependent manner and up to 14 days after uptake enabling generation of a calibration curve of the dose-residue relationship. Et-IPA was present in common vole blood for at least 14 days, but there was dissipation by 33-37% depending on dose. Et-IPA meets many criteria for an "ideal" quantitative bait marker for use in future field studies on common voles and possibly other small mammal species.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis by LC/ESI-MS of iophenoxic acid derivatives and evaluation as markers of oral baits to deliver pharmaceuticals to wildlife.

Iophenoxic acid and its derivatives (methyl, ethyl, and propyl) are organic chemicals used as markers in baiting campaigns to deliver vaccines, pharmaceuticals, contraceptives or poisons to wildlife. In this study we develop a method of detection of IPA derivatives by LC/ESI-MS (using butyl-IPA as internal standard) obtaining a limit of detection and quantification in wild boar (Sus scrofa) serum of 0.037 microg/ml and 0.123 microg/ml, respectively. The average recovery of IPA derivatives was 88% at levels >0.2 microg/ml, with coefficients of variation <15%. Wild boars in captivity were orally treated with 5 mg/kg b.w. (three adults) or 15 mg/kg b.w (two piglets and three adults) of methyl-, ethyl- and propyl-IPA and the serum levels of these were monitored during 18 months after dosing. Ethyl- and propyl-IPA were detected up to 18 months after a single oral dose in wild boar, especially at 15 mg/kg. Methyl-IPA was detected until 9 months after dosing. Half-lives of methyl-, ethyl- and propyl-IPA were (mean+/-SD) 41+/-5, 183+/-85 and 165+/-45 days, respectively. One control piglet not exposed to IPA, but housed in the same facility than treated animals showed detectable IPA levels in serum. Piglets born from mothers exposed to marked baits also showed detectable IPA levels in serum. The high persistence of Et- and Pr-IPA must be considered in the field trials, because the presence of the product at low levels in one animal may not reflect a real ingestion of the marked bait.

Liquid chromatography-electrospray ionization mass spectrometry for direct identification and quantification of iophenoxic acid in serum.

A liquid chromatographic-electrospray ionization mass spectrometric technique was developed for direct quantitation of iophenoxic acid (IA) in serum. IA was spiked into canine, feline, bovine, equine, and porcine sera, extracted, and quantified using negative ion monitoring following chromatographic separation on a Luna C18(2) 3 microm (100 mm x 2.1mm) reversed-phase column. The limit of detection was 25 ng/mL and the limit of quantification was 50 ng/mL. Inter- and intra-assay accuracy (86-113% and 87-115%, respectively) and precision (1.8-7.7%) were calculated. Analysis of serum collected from feral pigs, raccoons, and opossums following ingestion of IA-marked baits confirmed the appropriateness of this method for bait acceptance studies.

High-performance liquid chromatographic determination of iophenoxic acid in serum.

Iophenoxic acid (IPA), a marker used to investigate the feeding behaviour of bait-consuming animals has previously been indirectly determined by measuring protein-bound iodine levels in serum or plasma. For the first time a method is reported for the direct determination of IPA in biological fluids. IPA was determined in de-proteinized serum by high-performance liquid chromatography (HPLC) on a C18 column with a mobile phase of acetonitrile-water. Isocratic and gradient systems are described with limits of detection of 0.2 microgram/ml (isocratic) and 0.05 microgram/ml (gradient). Recoveries from fox serum were 85% at 0.5 microgram/ml, 95% at 5 micrograms/ml and 91% at 50 micrograms/ml.

Rapid assay of iopanoic acid in dog plasma using a Hisep column.

A rapid method for the determination of iopanoic acid (IOP) in dog plasma utilizing a Hisep column was developed. A mobile phase of 12% methanol, 88% 0.05 M phosphate buffer pH 3.4 yielded a k' of 8.5 with no interference from proteins present in plasma. Recoveries of IOP from spiked plasma ranged from 97% to 103% at 270 mumol/L and 1.75 mmol/L respectively. Replication was +/- 2.8% at 1.75 mmol/L and +/- 6% at 21 mumol/L. A method utilizing 2,4,6-triiodobenzoic acid as internal standard was also developed for comparison.

Concentration of 99mTc-HIDA and iopanoic glucuronide by the gallbladder.

To determine the ability of the gallbladder to concentrate Technetium 99mHIDA, both HIDA and iopanoic glucuronide were introduced directly into ligated gallbladders of anesthetized dogs. Serial bile samples were drawn to measure the concentration of each agent over a three hour period. Both biliary imaging agents were concentrated by the normal gallbladder. The results suggest that 99mTc-HIDA may be useful to study the gallbladder mucosal function of concentrating capacity in patients at risk for kidney damage by the standard cholecystographic agents like iopanoic acid.

Effects of anesthetics on the hepatic metabolism and biliary secretion of lopanoic acid enantiomers in rat.

The pharmacokinetics and metabolism of each iopanoate (IOP) enantiomer were studied in male rats anesthetized with urethane, pentobarbital or ether. The only metabolite of IOP that could be detected in bile and plasma was iopanoate glucuronide (IOP-G). Ether and pentobarbital significantly depressed the biliary secretion of each enantiomer as compared with urethane. The anesthetics did not affect differentially the plasma to liver concentration ratio of IOP or the relationship between the hepatic content of IOP-G and the biliary secretion rate of IOP-G for either enantiomer of IOP. At the end of 60 min, the total biliary secretion of (+)-IOP-G was significantly greater than that of (-)-IOP-G in the presence of each anesthetic agent. There was no apparent difference between (+)- and (-)-IOP with respect to hepatic accumulation. There were marked differences between the enantiomers with respect to the hepatic content of IOP-G. At the termination of the experiments, the relationship between the liver content of IOP-G and biliary secretion of IOP-G was not linear. Below a liver IOP-G content of approximately 0.12 mumol/g, the biliary secretion of (-)-IOP-G exceeded that of (+)-IOP-G. Above a liver IOP-G content of 0.12 mumol/g, the biliary secretion of (+)-IOP-G exceeded that of (-)-IOP-G. These data suggest a stereoselectivity with respect to the biliary secretion of IOP enantiomers.

Oral cholecystography: osmotic activity of iopanoic glucuronide in bile.

The cholecystographic agent iopanoic acid (Telepaque) is excreted in high concentrations in the bile as the glucuronide, but produces little or no choleresis. In order to determine the osmotic characteristics of the excretory product, bile was collected from treated and untreated anesthetized dogs and the major solute concentrations and biliary osmolality were determined. After iopanoic acid, there was no change in biliary osmolality or in chloride or bicarbonate concentration. However, the calculated osmolality exceeded the measured osmolality to a significant degree. The data suggest that iopanoic glucuronide, like bile salts, is osmotically inactive because of micelle formation. From physiological considerations, low osmotic activity of the excreted contrast agent might enhance radiographic quality.

Effect of choleretics on biliary and urinary excretion of cholecystographic agents.

Two choleretic agents, taurocholate and cinchophen, were studied in the dog to determine their effect on both the biliary and urinary excretion of two related cholecystographic agents, iopanoic acid and iophenoxic acid. Liver concentrations were determined on biopsies. Trace amounts of radioactively labeled compounds were used for measurements. The effect of the choleretic agent was specific for the cholecystographic compound. Taurocholate increased both biliary and urinary excretion of iopanoic acid and decreased its intrahepatic level, but had no effect on iophenoxic acid. Cinchophen had no effect on iopanoic acid but increased both biliary and urinary excretion of iophenoxic acid. When examined against a pre-existing high rate of bile flow, the active choleretic increased, rather than decreased, the biliary concentration of the cholecystographic compound. The increased excretion in both bile and urine cannot be attributed simply to the lessened concentration gradient from plasma to bile, as proposed by others. The data are consistent with a direct action of the choleretic agent on hepatic mechanisms, either to decrease intracellular binding or to alter the characteristics of transport.

Biliary and urinary excretion of iopanoic acid in the dog.

The urinary and biliary excretion of iopanonic acid was studied in anesthetized dogs infused with sodium iopanoate and trace amounts of 125I-iopanoic acid to provide a wide range of plasma concentrations. Samples of kidney and liver were also analyzed for total radioactivity. The sole excretory product in urine and bile was the glucuronide conjugate. The secretory biliary Tm for for iopanoic acid varied between experiments. Biliary concentration far exceeded that of plasma and liver/plasma ratios were all greater than unity. The fractional rate of urinary excretion varied 1-80% and was strongly correlated with hepatic function, e.g. the rate of bile flow. The data are interpreted to indicate that, depending on the rate of bile flow, the glucuronide formed in the liver is partioned between bile and blood, the latter for urinary excretion. This mechanism is discussed in relation to cholecystographic visualization and to the problem of nephrotoxicity.