The effect of P-glycoprotein on methadone hydrochloride flux in equine intestinal mucosa.

Methadone is an effective analgesic opioid that may have a place for the treatment of pain in horses. However, its absorption seems to be impaired by the presence of a transmembrane protein, P-glycoprotein, present in different tissues including the small intestine in other species. This study aims to determine the effect of the P-glycoprotein on methadone flux in the equine intestinal mucosa, as an indicator of in vivo drug absorption. Jejunum tissues from five horses were placed into the Ussing chambers and exposed to methadone solution in the presence or absence of Rhodamine 123 or verapamil. Electrical measurements demonstrated tissue viability for 120 min, and the flux of methadone across the jejunal membrane (mucosal to submucosal direction) was calculated based on the relative drug concentration measured by ELISA. The flux of methadone was significantly higher only in the presence of verapamil. P-glycoprotein was immunolocalized in the apical membrane of the jejunal epithelial cells (enterocytes), mainly located in the tip of the villi compared to cells of the crypts. P-glycoprotein is present in the equine jejunum and may possibly mediate the intestinal transport of methadone. This study suggests that P-glycoprotein may play a role in the poor intestinal absorption of methadone in vivo.

Performance of Standardized Relative CBV for Quantifying Regional Histologic Tumor Burden in Recurrent High-Grade Glioma: Comparison against Normalized Relative CBV Using Image-Localized Stereotactic Biopsies.

BACKGROUND AND PURPOSE: Perfusion MR imaging measures of relative CBV can distinguish recurrent tumor from posttreatment radiation effects in high-grade gliomas. Currently, relative CBV measurement requires normalization based on user-defined reference tissues. A recently proposed method of relative CBV standardization eliminates the need for user input. This study compares the predictive performance of relative CBV standardization against relative CBV normalization for quantifying recurrent tumor burden in high-grade gliomas relative to posttreatment radiation effects. MATERIALS AND METHODS: We recruited 38 previously treated patients with high-grade gliomas (World Health Organization grades III or IV) undergoing surgical re-resection for new contrast-enhancing lesions concerning for recurrent tumor versus posttreatment radiation effects. We recovered 112 image-localized biopsies and quantified the percentage of histologic tumor content versus posttreatment radiation effects for each sample. We measured spatially matched normalized and standardized relative CBV metrics (mean, median) and fractional tumor burden for each biopsy. We compared relative CBV performance to predict tumor content, including the Pearson correlation (r), against histologic tumor content (0%-100%) and the receiver operating characteristic area under the curve for predicting high-versus-low tumor content using binary histologic cutoffs (≥50%; ≥80% tumor). RESULTS: Across relative CBV metrics, fractional tumor burden showed the highest correlations with tumor content (0%-100%) for normalized (r = 0.63, P < .001) and standardized (r = 0.66, P < .001) values. With binary cutoffs (ie, ≥50%; ≥80% tumor), predictive accuracies were similar for both standardized and normalized metrics and across relative CBV metrics. Median relative CBV achieved the highest area under the curve (normalized = 0.87, standardized = 0.86) for predicting ≥50% tumor, while fractional tumor burden achieved the highest area under the curve (normalized = 0.77, standardized = 0.80) for predicting ≥80% tumor. CONCLUSIONS: Standardization of relative CBV achieves similar performance compared with normalized relative CBV and offers an important step toward workflow optimization and consensus methodology.

Pharmacokinetics of the injectable formulation of methadone hydrochloride administered orally in horses.

Methadone hydrochloride is a synthetic mu-opioid receptor agonist with potent analgesic properties. Oral methadone has been successfully used in human medicine and may overcome some limitations of other analgesics in equine species for producing analgesia with minimal adverse effects. However, there are no studies describing the pharmacokinetics (PK) of oral opioids in horses. The aim of this study was to describe the PK of orally administered methadone (0.1, 0.2 and 0.4 mg/kg) and physical effects in 12 healthy adult horses. Serum methadone concentrations were measured by gas chromatography/mass spectrometry at predetermined time points for 24 h, and PK parameters were estimated using a noncompartmental model. Physical effects were observed and recorded by experienced clinicians. No drug toxicity, behavioural or adverse effects were observed in the horses. The disposition of methadone followed first order elimination and a biphasic serum profile with rapid absorption and elimination phases. The PK profile of methadone was characterized by high clearance (Cl/F), small volume of distribution (V(d)/F) and short elimination half-life (t(1/2)). The mean of the estimated t(1/2) (SD) for each dose (0.1, 0.2 and 0.4 mg/kg) was 2.2 (35.6), 1.3 (46.1) and 1.5 (40.8), and the mean for the estimated C(max) (SD) was 33.9 (6.7), 127.9 (36.0) and 193.5 (65.8) respectively.

Optimization of Acquisition and Analysis Methods for Clinical Dynamic Susceptibility Contrast MRI Using a Population-Based Digital Reference Object.

BACKGROUND AND PURPOSE: The accuracy of DSC-MR imaging CBV maps in glioblastoma depends on acquisition and analysis protocols. Multisite protocol heterogeneity has challenged standardization initiatives due to the difficulties of in vivo validation. This study sought to compare the accuracy of routinely used protocols using a digital reference object. MATERIALS AND METHODS: The digital reference object consisted of approximately 10,000 simulated voxels recapitulating typical signal heterogeneity encountered in vivo. The influence of acquisition and postprocessing methods on CBV reliability was evaluated across 6912 parameter combinations, including contrast agent dosing schemes, pulse sequence parameters, field strengths, and postprocessing methods. Accuracy and precision were assessed using the concordance correlation coefficient and coefficient of variation. RESULTS: Across all parameter space, the optimal protocol included full-dose contrast agent preload and bolus, intermediate (60°) flip angle, 30-ms TE, and postprocessing with a leakage-correction algorithm (concordance correlation coefficient = 0.97, coefficient of variation = 6.6%). Protocols with no preload or fractional dose preload and bolus using these acquisition parameters were generally less robust. However, a protocol with no preload, full-dose bolus, and low (30°) flip angle performed very well (concordance correlation coefficient = 0.93, coefficient of variation = 8.7% at 1.5T and concordance correlation coefficient = 0.92, coefficient of variation = 8.2% at 3T). CONCLUSIONS: Schemes with full-dose preload and bolus maximize CBV accuracy and reduce variability, which could enable smaller sample sizes and more reliable detection of CBV changes in clinical trials. When a lower total contrast agent dose is desired, use of a low flip angle, no preload, and full-dose bolus protocol may provide an attractive alternative.

Optimization of DSC MRI Echo Times for CBV Measurements Using Error Analysis in a Pilot Study of High-Grade Gliomas.

BACKGROUND AND PURPOSE: The optimal TE must be calculated to minimize the variance in CBV measurements made with DSC MR imaging. Simulations can be used to determine the influence of the TE on CBV, but they may not adequately recapitulate the in vivo heterogeneity of precontrast T2*, contrast agent kinetics, and the biophysical basis of contrast agent-induced T2* changes. The purpose of this study was to combine quantitative multiecho DSC MRI T2* time curves with error analysis in order to compute the optimal TE for a traditional single-echo acquisition. MATERIALS AND METHODS: Eleven subjects with high-grade gliomas were scanned at 3T with a dual-echo DSC MR imaging sequence to quantify contrast agent-induced T2* changes in this retrospective study. Optimized TEs were calculated with propagation of error analysis for high-grade glial tumors, normal-appearing white matter, and arterial input function estimation. RESULTS: The optimal TE is a weighted average of the T2* values that occur as a contrast agent bolus transverses a voxel. The mean optimal TEs were 30.0 ± 7.4 ms for high-grade glial tumors, 36.3 ± 4.6 ms for normal-appearing white matter, and 11.8 ± 1.4 ms for arterial input function estimation (repeated-measures ANOVA, P < .001). CONCLUSIONS: Greater heterogeneity was observed in the optimal TE values for high-grade gliomas, and mean values of all 3 ROIs were statistically significant. The optimal TE for the arterial input function estimation is much shorter; this finding implies that quantitative DSC MR imaging acquisitions would benefit from multiecho acquisitions. In the case of a single-echo acquisition, the optimal TE prescribed should be 30-35 ms (without a preload) and 20-30 ms (with a standard full-dose preload).

Improving Perfusion Measurement in DSC-MR Imaging with Multiecho Information for Arterial Input Function Determination.

BACKGROUND AND PURPOSE: Clinical measurements of cerebral perfusion have been increasingly performed with multiecho dynamic susceptibility contrast-MR imaging techniques due to their ability to remove confounding T1 effects of contrast agent extravasation from perfusion quantification. However, to this point, the extra information provided by multiecho techniques has not been used to improve the process of estimating the arterial input function, which is critical to accurate perfusion quantification. The purpose of this study is to investigate methods by which multiecho DSC-MRI data can be used to automatically avoid voxels whose signal decreases to the level of noise when calculating the arterial input function. MATERIALS AND METHODS: Here we compare postprocessing strategies for clinical multiecho DSC-MR imaging data to test whether arterial input function measures could be improved by automatically identifying and removing voxels exhibiting signal attenuation (truncation) artifacts. RESULTS: In a clinical pediatric population, we found that the Pearson correlation coefficient between ΔR2* time-series calculated from each TE individually was a valuable criterion for automated estimation of the arterial input function, resulting in higher peak arterial input function values while maintaining smooth and reliable arterial input function shapes. CONCLUSIONS: This work is the first to demonstrate that multiecho information may be useful in clinically important automatic arterial input function estimation because it can be used to improve automatic selection of voxels from which the arterial input function should be measured.

An NMR investigation of electron transfer in the copper-protein, plastocyanin.

1. The proton NMR spectra of oxidised and reduced French bean plastocyanin have been recorded on a 270 MHz pulsed spctrometer. 2. The spectrum of a mixture containing the protein in the paramagnetic Cu(II) and diamagnetic Cu(I) states is a superposition of the separate spectra. When ferrirate spectra. 3. The results show that self-exchange between Cu(II)- and Cu(I)-plastocyanin is slow on the NMR time scale (kex less than 2-10(4) M-1-s-1 at 50 degrees C), and that electron transfer in the presence of ferricyanide is rapid (k greater than 1-10(5) M-1-s-1).

Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses?

To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired laminitis. In addition, the effects of laminitis-inducing treatment have not been prevented or reversed by treatment with an MMP inhibitor or a vasoactive antagonist. It is possible that there is simultaneous activation of the vascular and enzymatic pathways and/or other inflammatory processes. Moreover, the third theory involving mechanical factors cannot be discounted simply because strong evidence for vascular and enzymatic changes exists. It is common for horses with severe musculoskeletal disease affecting weightbearing on a limb to develop laminitis in the contralateral limb. It remains to be determined what factors are responsible for initiation of laminitis in these individuals. Evidence has not been presented that precludes the possibility of coincident occurrence of vascular and enzymatic changes. In fact, many of the inflammatory mediators (e.g. interleukin-1beta) found in laminitic tissues can concurrently stimulate synthesis of vasoactive substances and activate MMPs. Because enzymatic action on proteins is largely dependent on the concentrations of proteins and enzyme, the enzymatic theory is not dependent upon increased delivery of enzymes via increased capillary flow. Likewise, because vascular changes can alter tissue function via increased capillary flow and oedema formation, the vascular theory is not dependent upon decreased capillary flow. It is true that naturally acquired laminitis is widely variable in severity and predisposing diseases. Therefore, most probably there are multiple mechanisms involved in the initiation and propagation of the pathophysiologic cascade(s) and, therefore, successful intervention will necessitate multiple treatment modalities.

Characterization of restricted diffusion in uni- and multi-lamellar vesicles using short distance iMQCs.

Improved understanding of the entrapment, transport, and release of drugs and small molecules within vesicles is important for drug delivery. Most methods rely on contrast agents or probe molecules; here, we propose a new MRI method to detect signal from water spins with restricted diffusion. This method is based on intermolecular double quantum coherences (iDQCs), which can probe the restricted diffusion characteristics at well-defined and tunable microscopic distance scales. By using an exceedingly short (and previously inaccessible) distance, the iDQC signal arises only from restricted diffusion spins and thereby provides a mechanism to directly image vesicle entrapment, transport, and release. Using uni- and multi-lamellar liposomes and polymersomes, we show how the composition, lamellar structure, vesicle size, and concentration affects the iDQC signal between coupled water spins at very short separation distances. The iDQC signal correlates well with conventional diffusion MRI and a proposed biexponential (multicompartmental) diffusion model. Finally, the iDQC signal was used to monitor dynamic changes in the lamellar structure as temperature-sensitive liposomes released their contents. These short distance iDQCs can probe the amount and diffusion of water entrapped in vesicles, which may be useful to further understand vesicle properties in materials science and drug delivery applications.

Evaluation of a wireless ambulatory capsule (SmartPill®) to measure gastrointestinal tract pH, luminal pressure and temperature, and transit time in ponies.

REASONS FOR PERFORMING STUDY: This study investigated the use of a wireless ambulatory capsule (WAC; SmartPill(®) pH.p GI Monitoring System) to determine WAC-gastric emptying time (GET) in ponies. OBJECTIVES: To measure WAC-GET and compare it to those findings with GET assessed by nuclear scintigraphy (S-GET). HYPOTHESIS: WAC-GET will be slower than S-GET, but will be significantly correlated. METHODS: Seven healthy adult mixed-breed pony mares were used in this study. Feed was withheld for 12 h prior to the WAC administration. After administration, a complete-feed diet was fed to allow the WAC to pass into the stomach. Luminal pH, temperature and pressure were collected by a modified receiver secured to the pony. Once the pH reached a value of ≥ 8.0, it was determined that gastric emptying had occurred, and ponies were fed grass hay. After 5 days, data were downloaded and analysed using proprietary software. During the second period of the study, after at least 2 weeks, 4 of the ponies underwent a standard S-GET test. RESULTS: The WAC was successfully administered, and data were collected from all ponies. The mean percentage of data packets collected by the receiver was 84.9 ± 3.51% (range 66.8-95.1%). Mean WAC-GET was 7.38 h (range 0.15-46.65 h). Mean gastric pH was 4.75 (range 2.07-6.99). Mean small intestinal transit time was 4.6 h. The mean pH for the small intestine was 8.0. The mean S-GET time (in hours) when 10% of the radioactive feed is present in the stomach (T-90%) was 2.3 h. The S-GET did not correlate significantly with the WAC-GET. CONCLUSIONS AND POTENTIAL RELEVANCE: The WAC was safely administered to ponies, and data were collected using a modified receiver. The WAC-GET varied considerably between ponies, but was ≤ 3 h in 5 of the 6 ponies. The WAC used in this study provided a noninvasive technique that produced novel information about the pony gastrointestinal tract, but owing to the substantial variability in GET values and long transit time it may not be a reliable clinical tool at this time.

A functional arginine residue in rabbit-muscle aldolase.

Rabbit muscle aldolase is irreversibly modified by the arginine-selective alpha-dicarbonyl, phenylglyoxal, loss of activity correlating with the unique modifications of one arginine residue per subunit, as determined by amino acid analysis, and (7-14C)phenylglyoxal incorporation. The affinity of the modified enzyme for dihydroxyacetone phosphate is significantly reduced while substantial protection against inactivation is afforded by fructose 1,6-disphosphate, dihydroxyacetone phosphate or phosphate ion. The nature of the substrate C-1 phosphate binding site in this enzyme is discussed in the light of these and other results.

Metal-replacement studies in Bacillus stearothermophilus aldolase and a comparison of the mechanisms of class I and class II aldolases.

A comparison of the product-inhibition patterns during cleavage of D-fructose 1,6-diphosphate by aldolases from yeast, rabbit muscle and Bacillus stearothermophilus shows an ordered reaction sequence for all three enzymes, with dihydroxyacetone phosphate the last-leaving product. Addition of Zn2+, Co2+, Fe2+, Mn2+ or Cd2+ ions to the inactive apo-(Bacillus stearothermophilus aldolase) restores activity to different extents, whereas Ni2+, Mg2+ or Cu2+ ions have no effect. The cleavage activity of this aldolase is not enhanced by added K+ ion. The effects of metal replacement on thermal stability, Km and Vmax. are given and the possible role of the metal is discussed in the light of these results.