Additional studies on nicotine exposure in horses: Accurate quantification and elimination profiles of potential biomarkers in plasma and urine.

RATIONALE: For the purpose of doping control, this is the first report of accurate quantification of four critical structural isomers of nicotine metabolites (trans-3'-hydroxycotinine, cis-3'-hydroxycotinine, 5'-hydroxycotinine, and N'-hydroxymethylnorcotinine) in equine plasma and urine for the establishment of their elimination profiles. Besides, the pharmacokinetic studies of trans-3'-hydroxycotinine and N'-hydroxymethylnorcotinine in equine plasma and urine are also presented for the first time. METHODS: The accurate quantification methods of the aforementioned four structural isomers in horse plasma and urine were successfully developed and validated using the solid-phase extractions followed by liquid chromatography/tandem mass spectrometry analysis. Baseline chromatographic separation was achieved to completely differentiate these isomers, which shared the same selected reaction monitoring transition. Such methods were applied to post-administration samples obtained from the nicotine and tobacco leaf administration studies for the establishment of pharmacokinetic profiles. RESULTS: N'-Hydroxymethylnorcotinine could be quantified for the longest period, ranging from 48 to 72 h in plasma and 96 h in urine after a single administration of 250 mg of nicotine and an equivalent amount of nicotine in tobacco leaves. In terms of detection, both N'-hydroxymethylnorcotinine and trans-3'-hydroxycotinine could be detected up to the last sample collection time point (96 h), indicating that they are the most appropriate biomarkers for nicotine exposure. CONCLUSIONS: N'-Hydroxymethylnorcotinine and trans-3'-hydroxycotinine were detected longest in plasma and urine samples after both nicotine and tobacco leaf administrations, and N'-hydroxymethylnorcotinine was deemed most appropriate as a monitoring target due to its relatively higher abundance and slower elimination rate. These two biomarkers could also be used to differentiate sample contamination by tobacco products and genuine nicotine exposure to horse regardless of intentionality.

Detection and longitudinal distribution of GW1516 and its metabolites in equine hair for doping control using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: GW1516 is a peroxisome proliferator-activated receptor-δ (PPAR-δ) agonist that is banned in horseracing and equestrian sports. Long-term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. METHODS: Mane hairs were divided into three segments (0-7, 7-15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using a Q-Exactive instrument. This method was successfully validated and applied to post-administration samples to confirm the presence of GW1516 and its metabolites and estimate the uptake amounts of GW1516. RESULTS: After administration of 150 mg of GW1516 to a thoroughbred mare, GW1516 was detected in one of two segments of all mane hairs, and four metabolites, namely GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), and 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), were also identified. The longitudinal distribution analysis results showed that the maximum uptake of GW1516 into hair (approximately 0.05 pg/mg) was observed at around 13 weeks post-administration and GW1516 could be detected and confirmed up to 6 months post-administration. CONCLUSIONS: The parent drug GW1516 was identified as the most appropriate monitoring target in equine hair for controlling its misuse in horses. The use of hair analysis could extend the detection time of GW1516 to at least 6 months after the administration of 150 mg of GW1516 to a thoroughbred mare.

Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry for doping control.

RATIONALE: The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. METHODS: Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full-scan mass spectral data of pre- and post-administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine. RESULTS: GW1516 and its nine metabolites [including GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), methyl 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylate (MMTC), 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), and M1 to M4] were detected in post-administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post-administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine. CONCLUSIONS: For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.

Spore germination and ectomycorrhizae formation of Tricholoma matsutake on pine root systems with previously established ectomycorrhizae from a dikaryotic mycelial isolate of T. matsutake.

In vitro ectomycorrhizal synthesis of Tricholoma matsutake with host plants has been widely conducted to elucidate fungal symbiotic properties for future cultivation practices. Here, we report on the importance of basidiospore inocula for this fungus to provide ectomycorrhizal seedlings in vitro. Ectomycorrhizal pine seedlings synthesized in vitro with cultured mycelium of T. matsutake (isolate #45 or #84) in a 250-mL culture vessel (soil volume) were transplanted to a large 1-L culture vessel. Fresh basidiospores of this fungus were aseptically inoculated on the ectomycorrhizal root system. The ectomycorrhizal seedlings in the 1-L vessel were grown for 9 months, and some plants were further grown for 6 more months under non-aseptic conditions in 4.1-L jars. The ectomycorrhizal seedlings previously inoculated with isolate #84 in the 1-L vessel showed significant ectomycorrhizal biomass (mycorrhizal root length) after spore inoculation. The ectomycorrhizal seedlings in the 4.1-L vessel showed large shiro structures (> 10 cm in diameter). PCR amplification of intergenic spacer 1 of the rRNA gene and long terminal repeat retroelement of T. matsutake in ectomycorrhizal root tips in both the 1-L vessels and 4.1-L jars revealed the presence of amplicons of the previously inoculated culture isolate of T. matsutake and the new genet(s) that established via germination of the inoculated basidiospores. This is the first report that inoculated basidiospores of T. matsutake germinated and colonized the host root to generate ectomycorrhizae in vitro.

Doping control analysis of GW1516 in equine plasma using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry.

RATIONALE: GW1516 is a peroxisome proliferator-activated receptor-δ agonist in the class of hormones and metabolic modulators. The use of GW1516 is banned in both horseracing and equestrian competitions. To the best of our knowledge, this is the first metabolic study of GW1516 in horses. METHODS: After protein precipitation of pre- and post-administration plasma GW1516 samples, the supernatants were analyzed using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry to detect GW1516 and its metabolites. Monoisotopic ions of GW1516 and its metabolites were monitored from the full-scan mass spectral data of pre- and post-administration samples. Quantification methods were developed and validated to establish the elimination profiles of GW1516, its sulfoxide, and its sulfone in equine plasma. RESULTS: GW1516 and its four metabolites GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), and M1 were detected in post-administration plasma samples. GW1516 sulfoxide, GW1516 sulfone, and HMTT were identified by comparison with their respective reference standards whereas M1 was tentatively identified as 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid by mass spectral interpretation. GW1516 had the longest detection time in post-administration plasma. The elimination profiles of GW1516, its sulfoxide, and its sulfone in plasma were established. CONCLUSIONS: For the purpose of doping control, GW1516 is recommended as the target analyte to be monitored in equine plasma due to its long detection time (around 1 week) and the ready availability of its reference material.

Repeated fruiting of Japanese golden chanterelle in pot culture with host seedlings.

Yellow chanterelles are among the most popular wild edible ectomycorrhizal mushrooms worldwide. The representative European golden chanterelle, Cantharellus cibarius, has only once been reported to fruit under greenhouse conditions, due to the difficulty of establishing pure culture. Recently, we developed a new technique for establishing a pure culture of a Japanese golden chanterelle (Cantharellus anzutake), and conducted in vitro ectomycorrhizal synthesis using established strains and Pinus densiflora. Acclimated pine mycorrhizal seedlings colonized with C. anzutake in a pot system under laboratory conditions produced small but distinct basidiomata with developed basidiospores. C. anzutake mycorrhizae were established on Quercus serrata seedlings by inoculation of mycorrhizal root tips of the fungus synthesized on P. densiflora. A scaled-up C. anzutake-host system in larger pots (4 L soil volume) exhibited repeated fruiting at 20-24 °C under continuous light illumination at 150 µmol m-2 s-1 during a 2-year incubation period. Therefore, a C. anzutake cultivation trial is practical under controlled environmental conditions.

Quantification of horse plasma proteins altered by xylazine using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry.

In the doping tests currently used in horse racing, prohibited substances or their metabolites are usually directly detected in urine or blood samples. However, despite their lasting pharmaceutical effects, some prohibited substances are rapidly eliminated from horse urine and blood, making them difficult to detect. Therefore, new indirect biomarkers for doping, such as plasma proteins that are increased by the prohibited substances, have recently attracted much attention. Here, a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method was adopted for horse plasma proteomics analysis, in order to identify plasma proteins whose concentrations were altered in response to xylazine in Thoroughbred horses. Xylazine, which is rapidly absorbed and eliminated and has possibility of the change in the levels of plasma proteins, was selected as a model drug. Of the ten plasma proteins identified, four proteins, including three acute phase proteins (haptoglobin, ceruloplasmin, and α-2-macroglobulin-like), were significantly increased after xylazine administration. Therefore, our present approach might be useful in identifying indirect biomarkers of drug administration.

Modified signal-to-noise ratio in the liver using the background-to-lung activity ratio to assess image quality of whole-body 18F-fluorodeoxyglucose positron emission tomography.

The signal-to-noise ratio in the liver (SNR liver) is commonly used to assess the quality of positron emission tomography (PET) images; however, it is weakly correlated with visual assessments. Conversely, the noise equivalent count (NEC) density showed a strong correlation with visual assessment but did not consider the effects of image reconstruction conditions. Therefore, we propose a new indicator, the modified SNR liver, and plan to verify its usefulness by comparing it with conventional indicators. We retrospectively analyzed 103 patients who underwent whole-body PET/computed tomography (CT). Approximately 60 min after the intravenous injection of 18F-fluorodeoxyglucose (FDG), the participants were scanned for 2 min/bed. The SNR liver and NEC density were calculated according to the Japanese guidelines for oncology FDG-PET/CT. The modified SNR live was calculated by multiplying the background-to-lung activity ratio by the SNR liver. Patients were classified into groups based on body mass index (BMI) and visual scores. Subsequently, the relationships between these physical indicators, BMI, and visual scores were evaluated. Although the relationship between the modified SNR liver and BMI was inferior to that of NEC density and BMI, the modified SNR liver distinguished the BMI groups more clearly than the conventional SNR liver. Additionally, the modified SNR liver distinguished low visual scores from high scores more accurately than the conventional SNR liver and NEC density. Whether the modified SNR liver is more suitable than the NEC density remains equivocal; however, the modified SNR liver may be superior to the conventional SNR liver for image-quality assessment.

Phantom-Based Standardization Method for 123I-metaiodobenzylguanidine Heart-to-Mediastinum Ratio Validated by D-SPECT Versus Anger Camera.

Background: The 123I-metaiodobenzylguanidine heart-to-mediastinum ratios (HMRs) have been standardized between D-SPECT and Anger cameras in a small patient cohort using a phantom-based conversion method. This study aimed to determine the validity of this method and compare the diagnostic performance of the two cameras in a larger patient cohort. Methods: We retrospectively calculated HMRs from early and late anterior-planar equivalent and planar images acquired from 173 patients in 177 studies using D-SPECT and Anger cameras, respectively. The D-SPECT HMRs were cross-calibrated to an Anger camera using conversion coefficients based on previous phantom findings, then standardized to medium-energy general-purpose collimator conditions. Relationships between HMRs before and after corrections were investigated. Late HMRs were classified into four cardiac mortality risk groups and divided into two groups using a threshold of 2.2 to verify diagnostic performance concordance. Results: Correction improved linear regression lines and differences in HMRs among the groups. The overall ratios of perfect concordance were (134 [75.7%] of 177), and higher in groups with very low (49 [80.3%] of 61) and high (51 [86.4%] of 59) HMRs when the standardized HMR was classified according to cardiac mortality risk. That between the systems was the highest (164 [92.7%] of 177) when the HMR was divided by a threshold value of 2.2. Conclusions: Phantom-based conversion can standardize HMRs between D-SPECT and Anger cameras because the standardized HMR provided comparable diagnostic performance. Our findings indicated that this conversion could be applied to multicenter studies that include both D-SPECT and Anger cameras.

Beads phantom for evaluating heterogeneity of SUV on 18F-FDG PET images.

PURPOSE: This study aimed to develop a dedicated phantom using acrylic beads for texture analysis and to represent heterogeneous 18F-fluorodeoxyglucose (FDG) distributions in various acquisition periods. METHODS: Images of acrylic spherical beads with or without diameters of 5- and 10-mm representing heterogeneous and homogeneous 18F-FDG distribution in phantoms, respectively, were collected for 20 min in list mode. Phantom data were reconstructed using three-dimensional ordered subset expectation maximization with attenuation and scatter corrections, and the time-of-flight algorithm. The beads phantom images were acquired twice to evaluate the robustness of texture features. Thirty-one texture features were extracted, and the robustness of texture feature values was evaluated by calculating the percentage of coefficient of variation (%COV) and intraclass coefficient of correlation (ICC). Cross-correlation coefficients among texture feature values were clustered to classify the characteristics of these features. RESULTS: Heterogeneous 18F-FDG distribution was represented by the beads phantom images. The agreements of %COV between two measurements were acceptable (ICC ≥ 0.71). All texture features were classified into four groups. Among 31 texture features, 24 exhibited significant different values between phantoms with and without beads in 1-, 2-, 3-, 4-, 5-, 20-min image acquisitions. Whereas, the homogeneous and heterogeneous 18F-FDG distribution could not be discriminated by seven texture features: low gray-level run emphasis, high gray-level run emphasis, short-run low gray-level emphasis, low gray-level zone emphasis, high gray-level zone emphasis, short-zone low gray-level emphasis, and coarseness. CONCLUSIONS: We have developed the acrylic beads phantom for texture analysis that could represent heterogeneous 18F-FDG distributions in various acquisition periods. Most texture features could discriminate homogeneous and heterogeneous 18F-FDG distributions in the beads phantom images.

Comprehensive metabolic study of nicotine in equine plasma and urine using liquid chromatography/high-resolution mass spectrometry for the identification of unique biomarkers for doping control.

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves.

Identification of potential biomarkers in urine and plasma after consumption of tobacco product in horses.

The use of nicotine stimulants in horses is generally banned in horse racing and equestrian sports-accidental consumption of tobacco products is one of the possible causes of nicotine exposure in horses. The authors recently reported a comprehensive metabolic study of nicotine in equines, differentiating between nicotine exposure and sample contamination by means of a nicotine biomarker trans-3'-hydroxycotinine. To identify potential biomarkers for the differentiation of genuine nicotine administration and consumption of tobacco products, tobacco leaves (equivalent to 250 mg of nicotine) were nasoesophageally administered to three thoroughbred mares. Quantification methods of anatabine in plasma and urine were newly developed and validated and successfully applied to postadministration samples. Previously reported simultaneous quantification methods of eight target analytes including nicotine and its metabolites in plasma and urine were also applied to the samples. The results demonstrate that both trans-3'-hydroxycotinine and anatabine could be used as potential biomarkers in equine urine and plasma to indicate recent exposure to tobacco products in horses. As well, trans-3'-hydroxycotinine had the longest half-life as a detectable metabolite in urine and plasma. To our knowledge, this is the first report of a comprehensive study of tobacco product detection in horses.

Medication control of flunixin in racing horses: Possible detection times using Monte Carlo simulations.

BACKGROUND: For medication control in several jurisdictions, withdrawal time is the period of refrain from racing after drug administration. It is set by adding a safety period to an experimental detection time. However, there are no reports of statistical analyses of detection time for the determination of withdrawal time in flunixin meglumine-treated horses. OBJECTIVE: To analyse the population pharmacokinetics of flunixin in horses through the generation of a dataset for detection time statistical analysis and predictions via Monte Carlo simulation. STUDY DESIGN: Experimental study. METHODS: Drug plasma and urine concentrations following single intravenous administration of flunixin 1.1 mg/kg bodyweight (BW) in 10 horses and multiple administration of q 24 hours for 5 days in 10 horses were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Data were modelled using a nonlinear mixed effect model followed by Monte Carlo simulation. Irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were calculated using the Toutain approach. Detection times were obtained considering the time after the last administration for selected quantiles of 5000 hypothetical horses under the international screening limit (ISL) proposed by the International Federation of Horseracing Authorities (plasma: 1 ng/mL, urine; 100 ng/mL). RESULTS: For a regimen of 1.1 mg/kg BW q 24 hours, the IPC and IUC values were 2.0 and 73.0 ng/mL respectively. Detection times in plasma above the ISL for 90% of simulated horses were estimated as 74 hours after a single 1.1 mg/kg dose administration, 149 and 199 hours after multiple doses over 5 days at either 24- or 12-hour intervals respectively. Corresponding detection times in urine were 46, 68 and 104 hours respectively. MAIN LIMITATION: Only female horses were investigated. CONCLUSIONS: Statistical detection times for different flunixin meglumine regimens indicated a delay of detection time in plasma after multiple administrations under ISL.

Texture Feature Comparison Between Step-and-Shoot and Continuous-Bed-Motion 18F-FDG PET.

Our objective was to investigate the differences in texture features between step-and-shoot (SS) and continuous-bed-motion (CBM) imaging in phantom and clinical studies. Methods: A National Electrical Manufacturers Association body phantom was filled with 18F-FDG solution at a sphere-to-background ratio of 4:1. SS and CBM were performed using the same acquisition duration, and the data were reconstructed using 3-dimensional ordered-subset expectation maximization with time-of-flight algorithms. Texture features were extracted using the software LIFEx. A volume of interest was delineated on the 22-, 28-, and 37-mm spheres with a threshold of 42% of the maximum SUV. The voxel intensities were discretized using 2 resampling methods, namely a fixed bin size and a fixed bin number discretization. The discrete resampling values were set to 64 and 128. In total, 31 texture features were calculated with gray-level cooccurrence matrix (GLCM), gray-level run length matrix, neighborhood gray-level different matrix, and gray-level zone length matrix. The texture features of the SS and CBM images were compared for all settings using the paired t test and the coefficient of variation. In a clinical study, 27 lesions from 20 patients were examined using the same acquisition and image processing as were used during the phantom study. The percentage difference (%Diff) and correlation between the texture features from SS and CBM images were calculated to evaluate agreement between the 2 scanning techniques. Results: In the phantom study, the 11 features exhibited no significant difference between SS and CBM images, and the coefficient of variation was no more than 10%, depending on resampling conditions, whereas entropy and dissimilarity from GLCM fulfilled the criteria for all settings. In the clinical study, the entropy and dissimilarity from GLCM exhibited a low %Diff and excellent correlation in all resampling conditions. The %Diff of entropy was lower than that of dissimilarity. Conclusion: Differences between the texture features of SS and CBM images varied depending on the type of feature. Because entropy for GLCM exhibits minimal differences between SS and CBM images irrespective of resampling conditions, entropy may be the optimal feature to reduce the differences between the 2 scanning techniques.

Pharmacokinetics and tissue distribution of minocycline hydrochloride in horses.

OBJECTIVE: To determine the pharmacokinetics and tissue distribution of minocycline in horses. ANIMALS: 5 healthy Thoroughbred mares for the pharmacokinetic experiment and 6 healthy Thoroughbred mares for the tissue distribution experiment. PROCEDURES: Each mare was given 2.2 mg of minocycline hydrochloride/kg, IV. Blood samples were collected once before minocycline administration (0 hours) and 10 times within 48 hours after administration in the pharmacokinetics study, and 24 tissue samples were obtained at 0.5 and 3 hours in the distribution study. RESULTS: No adverse effects were observed in any of the mares after minocycline administration. The mean+/-SD elimination half-life was 7.70+/-1.91 hours. The total body clearance was 0.16+/-0.04 L/h/kg, and the volume of distribution at steady state was 1.53+/-0.09 L/kg. The percentage of plasma protein binding was 68.1+/-2.6%. Plasma concentration of free minocycline was 0.12 microg/mL at 12 hours. Minocycline was not detected in brain tissue, CSF or aqueous humor at 0.5 hours; however, it was found in all tissues, except in the aqueous humor, at 3 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Clearance of minocycline in healthy mares was greater than that reported for humans. For effective treatment of infections with common equine pathogens, it will be necessary to administer minocycline at a dosage of 2.2 mg/kg, IV, every 12 hours. This drug could be useful for infections in many tissues, including the CNS. The pharmacokinetic and tissue distribution data should aid in the appropriate use of minocycline in horses.

Presence of Antimicrobials in Postrace Samples in Japanese Thoroughbred Racing.

Ever since 'One Health' concept was introduced in early 2000s, judicious use of antimicrobials by veterinarians has become an issue of great concern. Recently, findings of anti-inflammatory effects in certain types of antimicrobials have raised a subject for discussion among racing authorities. Regulatory framework of antimicrobials in racing should be based on best interest of horse welfare and doping control perspective, but basic data on prevalence of antimicrobials are lacking. Analysis of 100 postrace urinary samples collected from 10 Japanese racecourses by targeting 21 antimicrobials using ultra performance liquid chromatography-tandem mass spectrometry resulted in detection of ceftiofur, cefalotin, cefalotin metabolite, dihydrostreptomycin, gentamicin, kanamycin, and oxytetracycline. Detection of antimicrobials critically important for resistance in human medicine was limited to a single sample. Oxytetracycline, which is known to possess anti-inflammatory effects, was detected in three samples. This may suggest the need for establishing a regulatory framework from doping control perspective and further studies to clarify pharmacologically relevant concentration of antimicrobials with such properties.

Accuracy of metabolic volume and total glycolysis among six threshold-based target segmentation algorithms.

OBJECTIVE: This study aimed to evaluate the accuracy of six threshold-based segmentation methods with different target-to-background ratios (TBR), images with different voxel sizes and image noise, in measuring metabolic volume (MV) and total glycolysis (TG). METHODS: A standard body phantom consisting of six spheres (inner diameters of 37, 28, 22, 17, 13, and 10 mm) was filled with 18F-FDG solution. The background radioactivity level was 2.65 kBq/mL, and the TBRs were 4 and 8. PET data were acquired for 30 min with list mode. PET data for 30 and 3 min were reconstructed with a three-dimensional ordered subset expectation maximization algorithm plus time-of-flight information with images with 2 and 4 mm isotropic voxels. The six methods examined were absolute standardized uptake value (SUV) of 2.5 (SUV2.5), 41%, 50%, adaptive 41%, and adaptive 50% thresholds of maximum SUV (Th41, Th50, ThA41, and ThA50, respectively); and the contrast-oriented algorithm (ThCOA). Segmented MV and TG were compared with the actual inner volume and expressed as percentages (%MVseg and %TGseg, respectively). In addition, the segmented MV was converted to the diameter, and the differences of it from the reference diameter were compared among six methods. RESULTS: The ThCOA method yielded the most accurate measurements of %MVseg and %TGseg; the difference between %MVseg or %TGseg and its reference were smaller than 10% in 30-min and 15% in 3-min images, but the segmented contour was almost the same as the reference diameter. Measurements with Th50 and ThCOA were highly accurate for both %MVseg and %TGseg in the large spheres, and the adaptive threshold methods, including ThA41, ThA50, and ThCOA, were also highly accurate in the small spheres. The voxel sizes affected the accuracy of %MVseg and %TGseg with a TBR of 4 in any threshold-based methods. CONCLUSIONS: Of the six threshold-based segmentation methods studied, ThCOA was the most accurate method for evaluating MV and TG and had only minor dependence on TBRs and sphere size. The small voxel sizes improved the variation of the accuracy in low TBR.

Comparison of image quality between step-and-shoot and continuous bed motion techniques in whole-body 18F-fluorodeoxyglucose positron emission tomography with the same acquisition duration.

OBJECTIVE: This study aimed to compare the qualities of whole-body positron emission tomography (PET) images acquired by the step-and-shoot (SS) and continuous bed motion (CBM) techniques with approximately the same acquisition duration, through phantom and clinical studies. METHODS: A body phantom with 10-37 mm spheres was filled with 18F-fluorodeoxyglucose (FDG) solution at a sphere-to-background radioactivity ratio of 4:1 and acquired by both techniques. Reconstructed images were evaluated by visual assessment, percentages of contrast (%Q H) and background variability (%N) in accordance with the Japanese guideline for oncology FDG-PET/computed tomography (CT). To evaluate the variability of the standardized uptake value (SUV), the coefficient of variation (CV) for both maximum SUV and peak SUV was examined. Both the SUV values were additionally compared with those of standard images acquired for 30 min, and their accuracy was evaluated by the %difference (%Diff). In the clinical study, whole-body 18F-FDG PET/CT images of 60 patients acquired by both techniques were compared for liver signal-to-noise ratio (SNRliver), CV at end planes, and both SUV values. RESULTS: In the phantom study, the visual assessment and %Q H values of the two techniques did not differ from each other. However, the %N values of the CBM technique were significantly higher than those of the SS technique. Additionally, the CV and %Diff for both SUV values in the CBM images tended to be slightly higher than those in SS images. In the clinical study, the SNRliver values of CBM images were significantly lower than those of SS images, although the CV at the end planes in CBM images was significantly lower than those in SS images. In the Bland-Altman analysis for both SUV values, the mean differences were close to 0, and most lesions exhibited SUVs within the limits of agreement. CONCLUSIONS: The CBM technique exhibited slightly lesser uniformity in the center plane than the SS technique. Additionally, in the phantom study, the CV and %Diff of SUV values in CBM images tended to be slightly higher than those of SS images. However, since these differences were subtle, they might be negligible in clinical settings.

Importance of Defect Detectability in Positron Emission Tomography Imaging of Abdominal Lesions.

OBJECTIVES: This study was designed to assess defect detectability in positron emission tomography (PET) imaging of abdominal lesions. METHODS: A National Electrical Manufactures Association International Electrotechnical Commission phantom was used. The simulated abdominal lesion was scanned for 10 min using dynamic list-mode acquisition method. Images, acquired with scan duration of 1-10 min, were reconstructed using VUE point HD and a 4.7 mm full-width at half-maximum (FWHM) Gaussian filter. Iteration-subset combinations of 2-16 and 2-32 were used. Visual and physical analyses were performed using the acquired images. To sequentially evaluate defect detectability in clinical settings, we examined two middle-aged male subjects. One had a liver cyst (approximately 10 mm in diameter) and the other suffered from pancreatic cancer with an inner defect region (approximately 9 mm in diameter). RESULTS: In the phantom study, at least 6 and 3 min acquisition durations were required to visualize 10 and 13 mm defect spheres, respectively. On the other hand, spheres with diameters ≥17 mm could be detected even if the acquisition duration was only 1 min. The visual scores were significantly correlated with background (BG) variability. In clinical settings, the liver cyst could be slightly visualized with an acquisition duration of 6 min, although image quality was suboptimal. For pancreatic cancer, the acquisition duration of 3 min was insufficient to clearly describe the defect region. CONCLUSION: The improvement of BG variability is the most important factor for enhancing lesion detection. Our clinical scan duration (3 min/bed) may not be suitable for the detection of small lesions or accurate tumor delineation since an acquisition duration of at least 6 min is required to visualize 10 mm lesions, regardless of reconstruction parameters. Improvements in defect detectability are important for radiation treatment planning and accurate PET-based diagnosis.

Nucleotide sequence of equine erythropoietin and characterization of region-specific antibodies.

OBJECTIVE: To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO). SAMPLE POPULATION: RNA and lysate extracted from renal tissues of an adult Thoroughbred. PROCEDURE: Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the eEPO and hEPO amino acid sequences to obtain antibodies specific for eEPO and hEPO. Specificity of the antibodies was tested against supernatant of homogenized equine kidney and recombinant hEPO (rhEPO) by use of western immunoblotting techniques. RESULTS: Analysis of the 1,181 bp in the nucleotide sequence revealed that eEPO was a residue of 192 amino acids. Similarity of eEPO with amino acid sequences of EPO from other species was 81.0% to 90.6%. Antibodies were specifically recognized by eEPO or rhEPO molecules. Anti-hEPO (161 to 165) antibody specifically recognized rhEPO. In contrast, anti-eEPO (133 to 144) antibody reacted with the equine kidney lysate. CONCLUSIONS AND CLINICAL RELEVANCE: We determined the cDNA and amino acid sequence of eEPO and developed region-specific antibodies that specifically recognized eEPO or rhEPO. These antibodies may be useful in distinguishing rhEPO from eEPO in a test to detect the misuse of rhEPO in racehorses.