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D S Watt

Publications and source records attributed to D S Watt.

At least 19 recordsLinked to original sources
PubMed1999

Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase.

Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate-binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP. Without activating light, GTPgammaBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPgammaBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+-chelate affinity chromatography and reversed-phase HPLC, photolabeled peptides located within or near the phosphate-binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPgammaBP primarily modified a peptide near the middle of the GDH sequence, Asn135-Lys143 and Glu290-Lys295. However, ATPgammaBP modified a single peptide corresponding to the sequence Met411-Arg419 near the C-terminal domain. Using these results and the data from the previously identified base-binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three-dimensional schematic model structure derived from the bacterial enzyme.

Adenosine Diphosphate↗
PubMed1996

Planarity of nitro-substituted phenothiazines.

The structures of three nitro-substituted phenothiazines [1,3,4-trifluoro-2-nitrophenothiazine, 10-(4-chlorobutyl)-1,3,4-trifluoro-2-nitrophenothiazine and 10-(4-chlorobutyl)-3-nitrophenothiazine] have been determined. The first of these red compounds forms infinite stacks in the solid state, in which donor and acceptor regions of the approximately planar molecules alternate. The molecules of the other two compounds, which have folded, or 'butterfly', conformations in the solid state, do not form stacks, presumably because the bulky chlorobutyl substituents cannot be accommodated. The very dark color of solid 3-nitrophenothiazine suggests the presence of extended molecular stacks, but crystals suitable for a structure determination could not be obtained.

Crystallization↗
PubMed1996

Identification of metabolites of azaperone in horse urine.

Two metabolites of the tranquilizer azaperone were extracted from alkalinized horse urine after treatment with beta-glucuronidase/sulfatase from limpets (Patella vulgata). The metabolites were identified by a combination of independent chemical synthesis and GC/MS and 1H NMR analysis. The metabolites were identified as 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanol, designated as 5'-hydroxy-azaperol, and 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanone, designated as 5'-hydroxyazaperone. A TLC screening test was developed for detecting both metabolites in basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The screening test was used to detect azaperone metabolites in extracts of horse urine collected for 24 h after intravenous administration of azaperone. The administration of azaperone to horses was confirmed by GC/MS identification of 5'-hydroxyazaperone and 5'-hydroxyazaperol from basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The extracted metabolites were treated with bis(trimethylsilyl)acetamide to produce trimethylsilyl (TMS) ether derivatives, and mass spectra and retention times were compared to those of the synthesized metabolites treated in the same manner.

Animals↗
PubMed1993

Synthesis and application of bidentate photoaffinity cross-linking reagents. Nucleotide photoaffinity probes with two photoactive groups.

Two "targeted bidentate" photoaffinity cross-linking reagents, the monoanhydride of 8-N3ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]8-N3ATP gamma BP) and the monoanhydride of 8-N3GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]8-N3GTP gamma BP), were developed for studying the inter- and intramolecular interactions of nucleotide-binding proteins. Experiments using these bidentate reagents with two photoactive groups led to specific cross-linking: [gamma-32P]8-N3GTP gamma BP and [gamma-32P]8-N3ATP gamma BP showed intersubunit cross-linking of glutamate dehydrogenase and [gamma-32P]8-N3GTP gamma BP appeared to cross-link the alpha- and beta-subunits of tubulin. The non-azido "monodentate" versions of these reagents, the monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]ATP gamma BP) and the monoanhydride of GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]GTP gamma BP), were also synthesized and characterized. The ability of these monodentate reagents with one photoactive group to serve as photoaffinity probes was established by photolabeling specifically the exchangeable GTP-binding domain of tubulin with [gamma-32P]GTP gamma BP and the ATP-binding domain of purified adenylate kinase and several nucleotide-binding proteins in human brain homogenate with [gamma-32P]ATP gamma BP.

Adenosine Triphosphate↗
PubMed1993

Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

Adenosine Diphosphate↗
PubMed1992

The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Prostaglandin photoaffinity probes: synthesis and binding affinity of C-18 substituted PGF2 alpha prostanoids bearing a perfluorinated aryl azide.

C-18 Phenoxy analogs of prostaglandin F2 alpha (PGF2 alpha) that possessed a perfluorinated aryl azide and an aryl iodide substituent were synthesized and evaluated as potential photoaffinity probes for PGF2 alpha. Prior studies indicated that only hydrophobic modifications in the omega-side chain of PGF2 alpha were compatible with high binding affinity, and this finding excluded the use of a hydroxyl-substituted C-18 phenoxy group as an activated aryl ring capable of radioiodination. Consequently, an alternate means of introducing the iodine substituent using an ipsosubstitution of a trimethylsilyl arene was developed. Although this strategy was successful from a synthetic perspective, the potential PGF2 alpha photoaffinity probe, (15S)-18-[3'-((4''-azido-2'',3'',5'',6''-tetrafluorophenyl)- methoxy) methyl-5'-iodophenoxy]-19,20-bisnorprostaglandin F2 alpha, exhibited only marginal competitive binding with [3H]-PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. The hydrophobic but bulky C-18 substituent was presumably incompatible with effective receptor binding.

Affinity Labels↗
PubMed1992

Prostaglandin photoaffinity probes: synthesis and binding affinity of aryl azide-substituted C-1 esters of prostaglandin F2 alpha.

In seeking prostaglandin F2 alpha (PGF2 alpha) photoaffinity probes possessing both an efficient, photoactive cross-linking substituent and a radiolabel of high specific activity, the synthesis and binding affinity of PGF2 alpha C-1 esters in which the alcohol component possessed either an aryl azide or a perfluorinated aryl azide was investigated. These derivatives showed great promise due to their ability to compete for the binding of [3H]-PGF2 alpha in both a luteal membrane binding assay and in a whole luteal cell binding assay. Identification of the C-1 site in PGF2 alpha as a site for modification of the PGF2 alpha molecule with photoactive alcohol derivatives represented a logical step toward the goal of developing a useful PGF2 alpha photoaffinity probe.

Affinity Labels↗
PubMed1991

The photochemistry of iodo, methyl and thiomethyl substituted aryl azides in toluene solution and frozen polycrystals.

The photolysis of para-methyl and para-thiomethylphenylazide at 77 K produces the corresponding triplet nitrenes which can be detected by electron paramagnetic resonance (EPR) spectroscopy. Photolysis of these azides in frozen toluene at 77 K leads to insertion of the nitrene into a benzylic C-H bond of the matrix in modest yields. Photolysis of iodinated aryl azides under these conditions does not produce triplet nitrenes that can be detected by EPR spectroscopy. In contrast to the para-methyl and para-thiomethyl substituted phenyl nitrenes, photo-induced coupling of iodo-substituted phenyl nitrenes to toluene proceeds in very poor yield.

Azides↗
PubMed1990

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

Affinity Labels↗
PubMed1990

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 1. Characterization of binding.

A novel, specific, and potent biological action of leukotriene C4 (LTC4) was demonstrated in the Chinese hamster lung fibroblast cell line V79A03 (V79 cells), namely the confirment of protection against subsequent gamma-irradiation. Consequently, studies were conducted to determine whether LTC4-conferred radioprotection could be attributed to a receptor-mediated phenomenon. Specific binding sites for leukotriene C4 (LTC4) were identified and characterized using intact V79 cells incubated at 4 degrees C in the presence of serine-borate, during which time conversion of LTC4 to LTD4 or LTE4 was undetectable. Binding was maximal in a broad region between pH 6.2 and 8.8. Ca2+, Mg2+, and Na+ were not required for binding, and binding was not altered by GTP, ATP, or cAMP, by leukotrienes B4, D4, or E4, or by the leukotriene end point antagonists LY 171883, FPL 55712, or Revlon 5901-5. Scatchard analyses and kinetic experiments indicated the presence of high-affinity [Kd = 2.5 +/- 0.63 nM, approximately 9.9 x 10(5) sites/cell] and low-affinity [Kd = 350 +/- 211 nM, approximately 2.7 x 10(6) sites/cell] binding sites. The observed binding characteristics of LTC4 to V79 cells are consistent with a receptor-mediated phenomenon. In a companion communication which follows this report, we report the subcellular distribution of LTC4 binding to V79 cells and demonstrate that this binding is unlikely to be attributed principally to interaction with glutathione-S-transferase.

Animals↗
PubMed1990

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 2. Subcellular distribution of binding and unlikely role of glutathione-S-transferase.

It was reported previously that radiation-induced cytotoxicity in V79A03 (V79) cells was attenuated by pretreatment of cells with leukotriene C4 (LTC4), leading us to determine that V79 cells possessed specific binding sites, with characteristics of receptors, for LTC4 (see the preceding, companion communication). Additional studies were conducted to determine the subcellular distribution and the chemical nature of the LTC4 binding site in V79 cells. Trypsin treatment of cells before LTC4 binding assays resulted in a 74% reduction in high-affinity binding. In tests to examine the subcellular location of LTC4 binding, plasma membrane and nuclear fractions were obtained from V79 cells. In contrast to Scatchard analyses of LTC4 binding to intact cells which were curvilinear, Scatchard analyses of nuclear and plasma membrane fractions were linear, indicative of the presence in these cellular substituents of low and high-affinity binding, respectively. To examine the nature of the high-affinity LTC4 binding sites, intact V79 cells were photolyzed with [3H]-LTC4 rendered photoactive by preincubation with N-hydroxysuccinimidyl-4-azidobenzoate. The cell-bound radioactivity migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of approximately 40 kdal. Five different commercial preparations of glutathione-S-transferase (GST), which has been implicated as a source of LTC4 "specific binding" in other cells, migrated in the same SDS-PAGE system with an apparent molecular weight of 20-24 kdal. Furthermore, preincubations of V79 cells with three antisera generated against GST had minimal effects upon subsequent LTC4 binding to intact cells. These data, taken together with the data from the preceding companion communication, suggest that the radioprotective effect of LTC4 upon V79 cells may be attributable to a receptor-mediated phenomenon which appears distinct from leukotriene binding to GST.

Animals↗
PubMed1989

Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F2 alpha receptor. Implications for development of a photoaffinity probe.

The capacity of structurally modified analogs of prostaglandin F2 alpha (PGF2 alpha) to inhibit binding of [3H]PGF2 alpha to receptors on ovine luteal cells was evaluated by radioreceptor assay using dispersed, viable, ovine luteal cells. Binding assays were conducted at pH 5.75, since binding to both high (Kd 17.4 +/- 2.3 nM) and low (Kd 409 +/- 166 nM) affinity sites was enhanced markedly at reduced pH. The capability to compete with [3H]PGF2 alpha for binding was evaluated for different prostaglandin analogs having modifications in the C-8 "upper" side-chain, in the cyclopentane ring, or in the C-12 "lower" side-chain. Prostaglandin J2 was a surprisingly potent competitor for binding to the PGF2 alpha receptor. Several phenyl-substituted analogs exhibited receptor-binding potency greater than or equal to native PGF2 alpha, while most other analogs had reduced capacity to compete with native PGF2 alpha for binding. Several 17-azidophenol PGF2 alpha analogs were synthesized and tested, but analogs having hydroxyl groups on the aryl ring had low affinity for receptors. However, 17-(4-azidophenyl)-18,19,20-trinor-PGF2 alpha as well as 17-(3-iodo-4-azidophenyl)-18,19,20-trinor-PGF2 alpha exhibited binding affinities that were approximately 10% of native PGF2 alpha, and the radioiodinated analogs of PGF2 alpha may be useful as probes of the PGF2 alpha receptor.

Affinity Labels↗
PubMed1989

Prostaglandin photoaffinity probes: synthesis and biological activity of azide-substituted 16-phenoxy- and 17-phenyl-PGF2 alpha prostaglandins.

The development of a prostaglandin PGF2 alpha photoaffinity probe led to the synthesis and biological evaluation of azide-substituted 17-phenyl-18,19,20-trinorprostaglandin F2 alpha and 16-phenoxy-17,18,19,20-tetranorprostaglandin F2 alpha derivatives. Two approaches for the preparation of iodinated versions of these prostaglandins were evaluated: (1) iodination of a phenyl azide bearing an activating hydroxyl group and (2) iodination of an aniline precursor to the phenyl azide group and subsequent conversion of the aniline to the phenyl azide. In the first approach, 17-(4-azido-2-hydroxyphenyl)-18,19,20-trinorprostaglandin F2 alpha, 16-(5-azido-3-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha, and 16-(4-azido-2-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha were prepared by using the Corey synthesis, but were biologically inactive presumably as a result of the hydrophilic phenolic hydroxyl group. In the second approach, the iodination of a 17-(4-aminophenyl)-18,19,20-trinorprostaglandin F2 alpha derivative delivered 17-(4-azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha, which exhibited competitive binding with natural [3H]PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. [125I]-17-(4-Azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha was utilized in a preliminary photoaffinity cross-linking experiment.

Affinity Labels↗
PubMed1989

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells.

A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks' buffered saline was added to cells at 4 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees C. After recovery from cryopreservation, cell viability and prostaglandin F2 alpha (PGF2 alpha) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2 (PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep. Cells retrieved from cryostorage were evaluated by studying PGF2 alpha binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2 alpha) of PGF2 alpha binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 +/- 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2 alpha and increasing amounts of unlabeled PGF2 alpha) and unfractionated cells, we detected additional binding sites of lower affinity (Kd = 409 +/- 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Immunoassay detection of drugs in racing horses. XI. ELISA and RIA detection of fentanyl, alfentanil, sufentanil and carfentanil in equine blood and urine.

We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test for sufentanil and a 125I radioimmunoassay test for alfentanil as part of a panel of pre- and post-race tests for narcotic analgesics in racing horses. Our sufentanil ELISA test detects sufentanil with an I-50 of about 0.5 ng/ml. The test is rapid and economical in that it can be read with an inexpensive spectrophotometer, or even by eye. The test readily detects the presence of sufentanil or its metabolites in equine blood and urine from 1 to 24 hours respectively after administration of therapeutic or sub-therapeutic doses of this drug. Our sufentanil assay also cross-reacts with fentanyl, the methylated analogs of fentanyl (designer fentanyls), and carfentanil and detected these drugs in urine for several hours after their administration to horses. It does not, however, cross-react significantly with alfentanil. We have also developed an 125I radioimmunoassay for alfentanil. This test allows detection of alfentanil in blood and urine of horses for up to 4 hours after administration of this drug. As such, these tests are capable of improving the quality and reducing the cost of pre-race and post-race testing for fentanyl, sufentanil, carfentanil and alfentanil and a number of their congeners in racing horses. Similarly, these tests are capable of screening for these drugs in human drug abuse monitoring.

Alfentanil↗
PubMed1988

Immunoassay detection of drugs in racing horses. VII. Detection of acepromazine in equine urine and blood by ELISA and PCFIA.

We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test and a particle concentration fluorescence immunoassay (PCFIA) test for acepromazine as part of a panel of pre- and post-race tests for illegal medications in racing horses. These tests are rapid, sensitive and economical and development of the tests occurred in less than seven months. The ELISA test detects acepromazine with an I-50 of about 150 pg/ml. In vivo, it readily detects the presence of acepromazine or its metabolites in equine blood and urine from 8 to 72 hours or longer, respectively, after administration of sub-therapeutic doses. In vitro, the ELISA test cross-reacts with analogs of acepromazine, suggesting that it will also detect the use of other phenothiazine tranquilizers. The PCFIA test detects acepromazine with an I-50 of about 10 ng/ml. When applied to pre-race screening of serum samples as part of the pre-race testing program at a midwestern racetrack, the PCFIA test detected a number of cases of acepromazine abuse. Screening of stored post-race urine samples from associated horses by the ELISA test 'flagged' numerous samples for acepromazine, suggesting a pattern of acepromazine abuse. To date about twenty of these acepromazine flagged samples have been confirmed positive on mass spectrometry. As such the ELISA and PCFIA tests described in this communication are capable of substantially improving the quality of pre- and post-race testing programs for phenothiazine tranquilizers in racing horses.

Acepromazine↗