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.

Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometer for the purpose of doping control.

IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.

Intraoperative photodynamic detection of normal parathyroid glands using 5-aminolevulinic acid.

OBJECTIVE: It is important to identify and save the normal parathyroid glands during head and neck surgery because of their role in regulating the blood calcium level, yet it is often difficult to localize normal parathyroid glands during surgery. Fluorescence-guided parathyroidectomy in patients with hyperparathyroidism has already proved useful. However, there are few reports of fluorescence-guided localization of normal parathyroid glands in humans. We investigated the utility of fluorescence-guided localization of normal parathyroid glands during thyroidectomy and completed a spectral fluorescence analysis of the accumulation of 5-aminolevulinic acid metabolites in the parathyroid glands. METHODS: Eight patients with benign thyroid disease and five with malignant thyroid tumors were given 20 mg/kg body weight of 5-aminolevulinic acid orally 5 hours before surgery. After the posterior surface of the thyroid gland was exposed and the recurrent laryngeal nerve was identified, we illuminated the area with a violet-blue light of 405 nm. Tissues showing red fluorescence were biopsied to analyze the spectral fluorescence. RESULTS: Under the violet-blue light, normal parathyroid glands showed red fluorescence, while the surrounding structures such as the thyroid gland, muscles, and fat remained nonfluorescent. The spectral peak was observed at 635 nm indicating 5-aminolevulinic acid metabolites. Histopathologically, the biopsied tissue corresponded to normal parathyroid glands. CONCLUSIONS: 5-Aminolevulinic acid is useful to localize the normal parathyroid glands during thyroid surgery in humans.