Improved method for production of antibodies against T-2 toxin and diacetoxyscirpenol in rabbits.

A new, improved approach for the production of antibodies against T-2 toxin and diacetoxyscirpenol (DAS) was developed. The method involves the use of immunogens which were prepared by conjugating O-carboxymethoxyl oxime (CMO) derivatives of both toxins to bovine serum albumin (BSA). Isomers a and b of CMO-T-2 toxin and isomer b of CMO-DAS were tested. Antibodies against both toxins were demonstrated as early as 4 weeks after immunization. a-CMO-T-2-BSA conjugate was a better immunogen than the b isomer, and the highest titers (6,000) were reached 14 weeks after immunization and one booster injection. Antibody titers for rabbits immunized with the b isomer of CMO-T-2 never reached more than 2,000. The specificity of antibodies obtained from rabbits after immunization with CMO-T-2-BSA was similar to that of hemisuccinate-T-2-BSA. Anti-b-T-2 antibodies had slightly higher cross-reactivity with H-T-2 toxin than did the antibody obtained from rabbits immunized with the conjugate of the a isomer. The relative cross-reactivities of anti-a-CMO-T-2 antibody with T-2, acetyl-T-2, H-T-2, T-2-triol, 3'-OH-T-2, and T-2 tetraol were 1, 4.5, 5.7, 250, 500, and 3,000, respectively. The relative cross-reactivities of anti-b-T-2 antibody with T-2, acetyl-T-2, H-T-2, and T-2 triol were 1, 2, 3, and 488, respectively. Antibodies against b-CMO-DAS showed a high degree of cross-reactivity with monoacetoxyscirpenols (MAS). The relative cross-reactivities of anti-B-DAS antibody with DAS, 4-MAS, 15-MAS, acetyl-deoxynivalenol, T-2-toxin, acetyl-T-2, and neosolaniol were 1, 4, 5, 76, 107, 147, and 266, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved method for production of antibodies against T-2 toxin and diacetoxyscirpenol in rabbits

A new, improved approach for the production of antibodies against T-2 toxin and diacetoxyscirpenol (DAS) was developed. The method involves the use of immunogens which were prepared by conjugating O-carboxymethoxyl oxime (CMO) derivatives of both toxins to bovine serum albumin (BSA). Isomers a and b of CMO-T-2 toxin and isomer b of CMO-DAS were tested. Antibodies against both toxins were demonstrated as early as 4 weeks after immunization. a-CMO-T-2-BSA conjugate was a better immunogen than the b isomer, and the highest titers (6,000) were reached 14 weeks after immunization and one booster injection. Antibody titers for rabbits immunized with the b isomer of CMO-T-2 never reached more than 2,000. The specificity of antibodies obtained from rabbits after immunization with CMO-T-2-BSA was similar to that of hemisuccinate-T-2-BSA. Anti-b-T-2 antibodies had slightly higher cross-reactivity with H-T-2 toxin than did the antibody obtained from rabbits immunized with the conjugate of the a isomer. The relative cross-reactivities of anti-a-CMO-T-2 antibody with T-2, acetyl-T-2, H-T-2, T-2-triol, 3'-OH-T-2, and T-2 tetraol were 1, 4.5, 5.7, 250, 500, and 3,000, respectively. The relative cross-reactivities of anti-b-T-2 antibody with T-2, acetyl-T-2, H-T-2, and T-2 triol were 1, 2, 3, and 488, respectively. Antibodies against b-CMO-DAS showed a high degree of cross-reactivity with monoacetoxyscirpenols (MAS). The relative cross-reactivities of anti-B-DAS antibody with DAS, 4-MAS, 15-MAS, acetyl-deoxynivalenol, T-2-toxin, acetyl-T-2, and neosolaniol were 1, 4, 5, 76, 107, 147, and 266, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of antibody against deoxyverrucarol.

Immunization of rabbits with deoxyverrucarol (DOVE) conjugated to bovine serum albumin resulted in antibodies bound with either tritiated DOVE or diacetoxyscirpenol (DAS), but not with T-2 toxin. The affinity of antibodies with DOVE was found to be much higher than with DAS. When [3H] DOVE was used as a marking ligand in the competitive radioimmunoassay, the concentrations causing 50% inhibition of binding radioactivities by unlabeled DOVE, verrucarol, verrucarin A, and 4-monoacetoxyscirpenol were found to be 0.32, 1,070, 9,500, and 10,000 ng per assay, respectively. T-2 toxin, 15-monoacetoxyscirpenol, and deoxynivalenol gave less than 20% inhibition at 10 micrograms per assay. However, when [3H] DAS was used as the marking ligand, the concentrations causing 50% inhibition by DOVE, DAS, and verrucarol were found to be in the 50 to 60 ng per assay range. The antibodies are thus highly specific to DOVE rather than a common trichothecene backbone. The possible use of this antiserum for assay of macrocyclic trichothecenes is discussed.     

Production and characterization of a generic antibody against group A trichothecenes

An antibody against group A trichothecenes was produced after immunization of rabbits with an immunogen prepared by conjugation of T-2 toxin to bovine albumin at the C-8 position. T-2 toxin was first converted to 3-acetylneosolaniol (3-Ac-NEOS) and then to its hemisuccinate (HS) before conjugation to the protein. The rabbits showed a quick immune response after immunization of the new conjugate. The antibody produced bound with tritiated T-2 toxin, T-2 tetraol tetraacetate, and diacetoxyscirpenol (DAS) and showed good cross-reactivities with most of the group A trichothecenes. The concentrations causing 50% inhibition of binding of 3H-T-2 toxin to the new antibody by unlabeled T-2, acetyl-T-2, 3'-OH-T-2, DAS, 3-Ac-NEOS-HS, 3'-OH-Ac-T-2, T-2 tetraol tetraacetate, iso-T-2, 3-Ac-NEOS, Ac-DAS, and 3,4,15-triacetyl-7-deoxynivalenol were found to be 0.34, 0.34, 0.6, 2.5, 4, 10, 18, 24, 100, 200, and 300 ng/assay, respectively; for HT-2, T-2 triol, and T-2 tetraol, the concentration was greater than 1000 ng/assay. Nivalenol, deoxynivalenol (DON), 15-acetyl-DON, and triacetyl-DON, did not inhibit the binding at 1000 ng/assay. The practical application of using this new antibody for radioimmunoassay (RIA) of trichothecene was tested by spiking T-2 toxin to corn. T-2 toxin was then extracted with acetone, subjected to a simple Sep-Pak C-18 reversed-phase treatment, and analyzed by RIA. The overall recovery for 18 samples spiked with 10 to 50 ppb of T-2 toxin was 94.22%.     

Production and characterization of antibodies against nivalenol tetraacetate

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.     

Production and characterization of antibodies against nivalenol tetraacetate.

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.     

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay. The relative cross-reactivity of the monoclonal antibody in the CI-ELISA with the related trichothecenes such as triacetoxyscirpenol, 15-monoacetoxyscirpenol, diacetylverrucarol, 4-monoacetoxyscirpenol and scirpentriol were found to be 1.8, 0.8, 0.15, 0.02 and less than 0.001, respectively. The trichothecenes verrucarol, T-2 toxin, T-2 tetraol, deoxynivalenol, 3-acetyldeoxynivalenol and trichothecin showed no cross-reactivity.     

Uptake and effect of praziquantel and the major human oxidative metabolite, 4-hydroxypraziquantel, by Schistosoma japonicum

After exposure to praziquantel in vitro at a concentration of 1 microgram/ml for 0.5-2 hr, amounts of praziquantel in Schistosoma japonicum varied from 2.1 +/- 1.2 to 3.7 +/- 1.6 ng/male worm and 1.3 +/- 1.2 to 2.2 +/- 1.5 ng/female worm during the time studied. At 30 micrograms/ml, praziquantel amounts were 11-33-fold higher. However, within 2 hr after removal from a medium containing 30 micrograms/ml praziquantel, 95% of the drug was released from the parasites. When S. japonicum worm pairs were incubated in vitro with 1, 10, and 30 micrograms/ml of 4-hydroxypraziquantel, the major human oxidative metabolite of praziquantel, 0.2 +/- 0.2, 3.8 +/- 1.3, and 7.4 +/- 1.3 ng/worm pair, respectively, were found after a 2-hr incubation. 15-30-fold lower than corresponding worm pair amounts of praziquantel. In vivo, when 4- or 5-wk S. japonicum-infected mice were treated orally with praziquantel (300 mg/kg), peak concentrations of praziquantel in plasma determined by high pressure liquid chromatography were 14.7 +/- 1.5 micrograms/ml (4-wk infection) and 16.7 +/- 2.8 micrograms/ml (5-wk infection) 15 min after treatment. Corresponding in vivo worm praziquantel amounts were 1.8 +/- 0.4 ng/male worm and 2.4 +/- 1.1 ng/female worm, respectively, in the 4-wk infection and 4.6 +/- 1.6 ng/male worm and 5.6 +/- 1.2 ng/female worm in the 5-wk infection. Peak plasma concentrations of 4-hydroxypraziquantel were similar but corresponding in vivo worm amounts were 1-20-fold lower, depending on the time after drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

A HPLC method for the analysis of germacrone in rabbit plasma and its application to a pharmacokinetic study of germacrone after administration of zedoary turmeric oil

A validated new, simple and highly sensitive reversed-phase HPLC method is developed for studying the pharmacokinetics of germacrone after intravenous administration of zedoary turmeric oil (ZTO) oil-in-water microemulsion. The method did not require a complex and expensive equipment. A high extraction recovery (>80%) of germacrone was obtained. Linear calibration curves obtained with the peak-area ratio (y) of germacrone to internal standard (tanshinoneIIA) versus drug concentration (x) were found to be linear between 8.08 and 808 ng/ml. The limit of quantitation was 8.08 ng/ml.The monitored compounds were completely separated from others in ZTO and from endogenous species in plasma by HPLC. Pharmacokinetic investigations were performed on 18 male rabbits after intravenous administration of ZTO microemulsion via the ear vein at germacrone doses of 3.2, 6.4 and 9.6 mg/kg. The plasma concentration-time data fit to a two-compartment intravenous model with a weight of 1/C(2) (C: germacrone concentration in plasma). Germacrone exhibited linear pharmacokinetics after intravenous administration of ZTO microemulsion to rabbits over the germacrone dose range 3.2-9.6 mg/ml.     

Determination of dehydroandrographolide succinate in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS): Method development,validation and clinical pharmacokinetic study

A LC–MS/MS method was developed and validated for the determination of dehydroandrographolide succinate (DAS), a traditional Chinese medicine derivative used for the treatment of pneumonia, upper respiratory tract infection, and chronic bronchitis. Following protein precipitation, DAS was detected by ion transition at m/z 531.2/99.0 in multiple reaction monitoring mode with negative electrospray ionization-tandem mass spectrometry. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 10 ng/mL in human plasma. Good linearity was maintained over the range of 10–5000 ng/mL, and the correlation coefficient was better than 0.99. The accuracy ranged from 95.3% to 113%, RSD from 0.928% to 6.47%, for the within- and between-run analysis at all QC levels. The recovery ranged from 85.5% to 93.4% and the matrix effect from 107% to 119%. No significant carryover and good stability were found during method validation. The developed method was successfully applied to the determination of DAS in human plasma in a pharmacokinetic study following intravenous infusion of potassium sodium DAS to nine healthy volunteers.     

Production of neosolaniol monoacetate by an undescribedFusarium species resemblingF camptoceras

Cultures of 12 South African isolates of an undescribedFusarium species resembling but distinct fromF camptoceras were analysed for the presence of diacetoxyscirpenol (DAS), neosolaniol monoacetate (NMA), and T-2 toxin, by capillary gas chromatography utilizing electron capture detection. No DAS or T-2 toxin could be detected in any of the cultures of the isolates. NMA was, however, detected in 10 of the 12 isolates at levels ranging from 310 to 2060 ng/g. The method used, was primarily developed for the determination of DAS and T-2 toxin in fungal cultures and grain samples but was found to be suitable for the coextraction of NMA at an average recovery of 80.8%, with a detection limit in the order of 100 ng/g. Supportive evidence for the presence of the NMA was obtained by capillary gas chromatography / mass spectrometry. Regarded as a relatively rare trichothecene, NMA has never been reported to occur naturally and has previously been shown to be produced by only a fewFusarium strains.     

Development of a radioimmunoassay for 15-HETE and its application to 15-HETE production by reticulocytes

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.     

Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.

A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex.     

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes.

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

Some endocrine traits of transgenic rabbits. II. Changes in hormone secretion and response of isolated ovarian tissue to FSH and ghrelin

In the present in vitro experiments we examined FSH- and ghrelin-induced changes in ovarian hormone secretion by transgenic rabbits. Fragments of ovaries isolated from adult transgenic (carrying mammary gland-specific mWAP-hFVIII gene) and non-transgenic rabbits from the same litter were cultured with and without FSH or ghrelin (both at 0, 1, 10 or 100 ng/ml medium). The secretion of progesterone (P4), estradiol (E2) and insulin-like growth factor I (IGF-I) was assessed by RIA. It was observed that ovaries isolated from transgenic rabbits secreted much less P4, E2 and IGF-I than the ovaries of non-transgenic animals. In control animals FSH reduced E2 (at doses 1-100 ng/ml medium) and IGF-I (at 1-100 ng/ml), but not P4 secretion, whereas ghrelin promoted P4 (at 1 ng/ml) and IGF-I (at 100 ng/ml), but not E2 output. In transgenic animals, the effects were reversed: FSH had a stimulatory effect on E2 (at 100 ng/ml) and ghrelin had an inhibitory effect on P4 (at 10 ng/ml). No differences in the pattern of influence of FSH on P4 and IGF-I and of ghrelin on E2 and IGF-I were found between control and transgenic animals. The present observations suggest that 1) both FSH and ghrelin are involved in rabbit ovarian hormone secretion, 2) transgenesis in rabbits is associated with a reduction in ovarian secretory activity, and 3) transgenesis can affect the response of ovarian cells to hormonal regulators.     

15-Lipoxygenase metabolites contribute to age-related reduction in acetylcholine-induced hypotension in rabbits

Arachidonic acid (AA) metabolites from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetylcholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4-4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 +/- 7.4 and -37.9 +/- 3.9%)- and 4 (-48.8 +/- 2.4 and -35.5 +/- 7.8%)-wk-old rabbits were higher than 8 (-30.0 +/- 2.8 and -26.6 +/- 4.4%)- and 16 (-36.7 +/- 3.5 and -27.3 +/- 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and N(omega)-nitro-l-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with l-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to -6.9 +/- 0.9%, by apamin alone to -19.5 +/- 1.4%, and by BW755C to -18.8 +/- 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs.