Biomedical subjects

J Boyles

Publications and source records attributed to J Boyles.

39 records · Page 3Linked to original sources

PubMedDate not supplied

Detection and confirmation of ractopamine and its metabolites in horse urine after Paylean administration.

We have investigated the detection, confirmation, and metabolism of the beta-adrenergic agonist ractopamine administered as Paylean to the horse. A Testing Components Corporation enzyme-linked imunosorbent assay (ELISA) kit for ractopamine displayed linear response between 1.0 and 100 ng/mL with an I-50 of 10 ng/mL and an effective screening limit of detection of 50 ng/mL. The kit was readily able to detect ractopamine equivalents in unhydrolyzed urine up to 24 h following a 300-mg oral dose. Gas chromatography-mass spectrometry (GC-MS) confirmation comprised glucuronidase treatment, solid-phase extraction, and trimethylsilyl derivatization, with selected-ion monitoring of ractopamine-tris(trimethylsilane) (TMS) m/z 267, 250, 179, and 502 ions. Quantitation was elaborated in comparison to a 445 Mw isoxsuprine-bis(TMS) internal standard monitored simultaneously. The instrumental limit of detection, defined as that number of ng on column for which signal-to-noise ratios for one or more diagnostic ions fell below a value of three, was 0.1 ng, corresponding to roughly 5 ng/mL in matrix. Based on the quantitation ions for ractopamine standards extracted from urine, standard curves showed a linear response for ractopamine concentrations between 10 and 100 ng/mL with a correlation coefficient r > 0.99, whereas standards in the concentration range of 10-1000 ng/mL were fit to a second-order regression curve with r > 0.99. The lower limit of detection for ractopamine in urine, defined as the lowest concentration at which the identity of ractopamine could be confirmed by comparison of diagnostic MS ion ratios, ranged between 25 and 50 ng/mL. Urine concentration of parent ractopamine 24 h post-dose was measured at 360 ng/mL by GC-MS after oral administration of 300 mg. Urinary metabolites were identified by electrospray ionization (+) tandem quadrupole mass spectrometry and were shown to include glucuronide, methyl, and mixed methyl-glucuronide conjugates. We also considered the possibility that an unusual conjugate added 113 amu to give an observed m/z 415 [M+H] species or two times 113 amu to give an m/z 528 [M+H] species with a daughter ion mass spectrum related to the previous one. Sulfate and mixed methyl-sulfate conjugates were revealed following glucuronidase treatment, suggesting that sulfation occurs in combination with glucuronidation. We noted a paired chromatographic peak phenomenon of apparent ractopamine metabolites appearing as doublets of equivalent intensity with nearly identical mass spectra on GC-MS and concluded that this phenomenon is consistent with Paylean being a mixture of RR, RS, SR, and SS diastereomers of ractopamine. The results suggest that ELISA-based screening followed by glucuronide hydrolysis, parent drug recovery, and TMS derivatization provide an effective pathway for detection and GC-MS confirmation of ractopamine in equine urine.

Animals↗

PubMedDate not supplied

Uptake of apolipoprotein E-containing high density lipoproteins by hepatic parenchymal cells.

Cholesterol-enriched, apolipoprotein E-containing high density lipoproteins (apo E HDLc), which were isolated from the plasma of cholesterol-fed dogs by using agarose column chromatography or ultracentrifugation, possessed essentially identical biochemical and metabolic characteristics. Radioiodinated (125I) apo E HDLc isolated by either method gave identical rates of clearance from the plasma, i.e., greater than 50% of the injected dose was cleared from the plasma within 5 to 10 minutes, principally by the liver. Detailed studies localizing apo E HDLc uptake to specific cell types within the liver were performed in both normal and cholesterol-fed rats. The validity of using the canine apo E HDLc in the rat was supported by observations of marked similarities in plasma clearance, i.e., a rapid acute phase of disappearance, and a near-quantitative hepatic uptake of lipoproteins in both species. Canine apo E HDLc (fluorescently labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine), which were injected into normal rats, appeared to be taken up primarily by parenchymal cells, as determined by fluorescence microscopy. Light microscopic autoradiography also revealed that the uptake of 125I apo E HDLc was principally carried out by parenchymal cells in rat liver. Likewise, the uptake of apo E HDLc by the liver of cholesterol-fed rats was extensively localized to parenchymal cells. An in situ, single-pass perfusion of a lobule of the liver of a normal dog with iodinated and fluorescently labeled apo E HDLc confirmed that the uptake of the lipoproteins was principally carried out by parenchymal cells. The plasma clearance of apo E HDLc by hepatic parenchymal cells, even in cholesterol-fed animals in which the apo B,E (LDL) receptors were markedly down-regulated (undetectable), suggests that the apo E receptor, presumably the remnant receptor, is localized in the parenchymal cells.

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PubMedDate not supplied

Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary.

Previous studies showed that perfused 125I-labeled low and high density lipoproteins (LDL, HDL) have affinity for specialized microvillar regions of luteal cells in hormone-primed, luteinized rat ovaries. In the current report, we re-examined the interaction of cholesterol-rich lipoproteins with these specialized plasma membrane regions using native lipoproteins visualized as discrete particles by standard electron microscopic techniques. In ovaries perfused with the various lipoproteins, spherical particles (varying in size from 12 to 28 nm depending on the particle used) were found over the surfaces of all luteal cells and filling up extensive "channel" space formed by the apposed plasma membranes of adjacent microvilli or cytoplasmic surfaces. Only 30% of these tissue-associated particles were removable after prolonged washing with perfused media or heparin. Few intact particles were found inside the cells, despite the fact that the lipoproteins induced a substantial hormone response by the ovary. To determine the total protein internalized by cells during the course of the experiments, parallel biochemical experiments were carried out with nonreleasable (14C-sucrose-coupled) human LDL. Of the total bound 14C-sucrose LDL, only 8.5% was degraded (trichloroacetic acid-soluble) and presumed internalized by the cells. Thus, while large numbers of cholesterol-rich lipoprotein particles interact with the luteal cell surface in specialized microvillar channels and elicit a progesterone response, relatively few intact lipoprotein particles appear to enter the cells to be degraded. We speculate that in the luteinized ovary, a large majority of the lipoprotein-cholesterol transfer occurs at the surface of the luteal cells, and that the membranes of the microvillar channels are involved in this process.

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