Ergonovine Research Articles

Safety and potential usefulness of sequential intracoronary acetylcholine and ergonovine administration for spasm provocation testing.

Although guidelines recommend intracoronary acetylcholine (ACh) and ergonovine (ER) provocation testing for diagnosis of vasospastic angina, the feasibility and safety of sequential (combined) use of both pharmacological agents during the same catheterization session remain unclear. In this study, we investigated the feasibility and safety of sequential intracoronary ACh and ER administration for coronary spasm provocation testing. The study included 235 patients who showed positive results on ACh and ER provocation testing. Initial intracoronary ACh administration was followed by ER administration for left coronary artery (LCA) spasm provocation testing. Subsequently, the right coronary artery (RCA) was subjected to sequential ACh and ER administration for provocation testing. The primary outcome of the study was the safety of sequential intracoronary ACh and ER provocation testing, which was assessed based on a composite of all-cause death, sustained ventricular tachycardia and fibrillation, and cardiogenic shock. Even in patients with negative results on sequential intracoronary ACh and ER provocation testing in the LCA and only ACh administration into the RCA, additional administration of ER into the RCA showed a positive provocation test result in 33 of 235 (14.0%) patients; three (1.3%) patients developed adverse effects (cardiogenic shock occurred in all cases) during LCA provocation testing. We observed no deaths attributable to spasm provocation testing. Sequential administration of intracoronary ACh and ER was associated with a relatively low major complication rate and may be safe and potentially useful for diagnosis of vasospastic angina.

Abstract 16552: Coronary Vasomotor Response to Intracoronary Ergonovine Provocation Test According to the Severity of an Insignificant Coronary Artery Fixed Stenosis

BACKGROUND: Coronary artery spasm (CAS) is a major cause of variant angina in patients with chest pain without significant coronary stenosis. However, whether there are differences in the incidence of CAS according to the severity of underlying coronary artery fixed stenosis is limited. Methods: A total of 608 patients without significant coronary artery disease (CAD) who underwent the ergonovine (ERG) provocation test between January 2018 and December 2021 were enrolled. Significant CAS was defined as having≥70% of temporary narrowing by ERG test. Patients were divided into three groups, based on the presence of coronary artery stenosis: None group (n=456), mild group (30-49%,n=114) and moderate group (50-69%,n=38). Results: In patients with chest pain without significant coronary artery stenosis, mild stenosis was 18.8%, and moderate stenosis was 6.3% in the population. The baseline characteristics of the patients were worse in the mild and moderate group than in the None group, including age, hypertension, diabetes, and smoking habit. In the ERG test, CAS occurred in 44.4% (270/608) of the overall population. The incidence of CAS of the mild (55.3%) and moderate (52.6%) groups were higher than the None group (41.0%). Also, in the case of CAS occurrence, focal CAS (Length<20mm) was more frequent in the mild (14.3%) and moderate (30%) groups than in the no group (8.6%). CONCLUSIONS: In patients with resting chest pain without severe coronary artery stenosis, CAS was observed in a high proportion of nearly half by ergonovine provocation test. Moreover, the mild to moderate coronary artery fixed stenosis showed higher incidence of CAS and focal CAS than patients without fixed stenosis.

Intracoronary ergonovine testing among 505 consecutive Japanese patients with angina-like chest pain and unobstructed coronary artery disease.

Intracoronary ergonovine (ER) testing is useful for the detection of epicardial spasm (ES) and coronary microvascular spasm (CMS). We retrospectively analyzed the incidence of ES and CMS in consecutive Japanese patients with unobstructed coronary artery disease. From January 1991 to February 2019, we performed intracoronary ER testing of 1196 patients. Among these patients, a total of 505 consecutive patients (207 women, mean age 64 ± 11years) who underwent first diagnostic angiography for suspected myocardial ischemia and had unobstructed coronary arteries (< 50%) were enrolled. Resting chest pain was reported by 229 patients, exertional chest pain was reported by 62 patients, exertional and resting chest pain was reported by 61 patients, and another chest symptom (not typical chest pain but suspected to be myocardial ischemia) was reported by 153 patients. ES was defined as ≥ 90% stenosis and usual chest symptoms and ischemic ECG changes, while CMS was defined as < 75% stenosis (no epicardial spasm) and usual chest symptoms and ischemic ECG changes. We performed intracoronary ER testing on both coronary arteries in 86% (432/505) of all subjects. Overall, ES was found in 82 patients (16%), whereas CMS was revealed in 12 patients (2%). In patients with ES, women made up 9%, and 70% of the patients had resting chest pain. In contrast, women composed 67% (8/12) of those with CMS, and 5 patients with CMS had another chest symptom. Ventricular fibrillation was observed in two patients who had sinus rhythm after thump version or cardiac resuscitation. However, we observed no irreversible complications during ER testing. CMS was recognized in only 2% of consecutive Japanese patients with unobstructed coronary artery disease by intracoronary ER testing, whereas ES was revealed in 16% of those patients. CMS was often observed in women.

PSII-24 Assessment of the contractility of bovine lateral saphenous and digital veins in response to ergot alkaloids

Abstract Decreased blood flow caused by vasoconstriction is a hallmark of ergot alkaloid exposure. Previous research has utilized the cranial branch of the lateral saphenous vein as a model to characterize the impact of ergot alkaloids on bovine peripheral vasculature. The digital vein is more distal and closer to the hoof where damage caused by vasoconstriction occurs and may be more sensitive to ergot alkaloids. Thus, the objective was to compare contractility of the bovine lateral saphenous vein and the digital vein to increasing concentrations of ergot alkaloids. Blood vessels were collected from Holstein steers at slaughter (n = 12; 551 ±29 kg), cleaned, cut into 2-mm cross-sections, and mounted in a multi-myograph. In the myograph blood vessels were submersed in continuously gassed (95% O2/5% CO2) Krebs-Henseleit buffer. Following a 1.5-hr tension equilibration, blood vessels were exposed to a reference dose of 1x10-4 M norepinephrine. Digital and lateral saphenous veins from each steer were then exposed to increasing concentrations of a-ergocryptine (ERP), ergotamine (ERT), ergocristine (ERS), ergocornine (ERO), ergonovine (ERN), tall fescue seed extract (EXT; dilutions based on measured ergovaline concentration), lysergic acid (LSA), and lysergol (LYS) were diluted to final concentrations for use in the tissue bath of 5x10-10 to 1x10-4 M. All resultant contractile response data were normalized as a percentage of the 1x10-4 M norepinephrine response and analyzed as randomized design for effects of vein and alkaloid concentration. The maximal contractile response induced by EXT, ERT, ERO, ERS, and ERP did not differ by vein type and EXT was the most vasoactive alkaloid. The response to ERN was greater for the saphenous than the digital vein (P = 0.03) and LYS and LSA did not differ (P &amp;gt;0.05). These data confirm that the cranial branch of lateral saphenous vein is a representative model of peripheral vasculature of the bovine hindlimb.

Pharmacological spasm provocation testing in 2500 patients: provoked spasm incidence, complications and cardiac events

Pharmacological spasm provocation tests such as acetylcholine (ACh) and ergonovine (ER) had been performed in the clinic. We retrospectively analyzed the incidence of provoked spasm, complications during testing and the cardiac events after these tests. From January 1991 and October 2018, we performed pharmacological spasm provocation tests in 2500 patients: 1810 ACh tests, 1232 ER tests, 542 both tests, and 310 ACh added after ER tests. ACh was injected in incremental doses of 20/50/100/200μg into the LCA and 20/50/80μg into the RCA. ER was administered as a total dose of 64μg into the LCA and 40μg into the RCA. When adding ACh after ER, the total dose was 50/80μg into the RCA and 100/200μg into the LCA. Positive spasm was defined as ≥ 90% stenosis and usual chest pain or ischemic ECG changes. Mean follow-up duration was 47.5 ± 29.9months. Overall, provoked positive spasm was found in 1095 patients (43.8%). The incidence of positive provoked spasm during ACh testing was significantly higher than that during other tests (ACh: 48.7% vs. ER: 28.9%, Both: 24%, ACh added after ER: 33.5%, p < 0.001). Multiple spasms were remarkably more frequent during ACh testing compared with the other 3 types of testing (ACh: 28.2% vs. ER: 7.4%, Both: 4.1%, ACh added after ER: 13.2%, p < 0.001). No death or acute myocardial infarction was observed, while major complications during ACh testing were significantly more frequent than during ER testing. Readmission due to recurrent angina pectoris in spasm-positive patients was remarkably more frequent than in spasm-negative patients. The incidence of sudden cardiac death, ventricular fibrillation, and acute coronary syndrome were not different between the spasm-positive and spasm-negative groups during the follow-up periods. We could perform all spasm provocation tests without any irreversible complications. All sequential spasm provocation tests were useful for documenting coronary spasm.

Clinical effect of ergometrine maleate injection combined with posterior pituitary injection in the treatment of postpartum hemorrhage

Objective To investigate the clinical effect of ergometrine maleate injection combined with posterior pituitary injection in the treatment of postpartum hemorrhage. Methods From April 2016 to July 2018, 68 patients with postpartum hemorrhage treated in the Maternal and Child Health Care Hospital of Tongxiang were randomly divided into two groups according to the random number table, with 34 patients in each group.The control group was treated with posterior pituitary injection.The observation group was treated with ergometrine maleate injection combined with posterior pituitary injection.The bleeding volume at different time points after treatment was compared between the two groups.The hemostasis time, the duration of uterine contraction, the extent of uterine decline, the duration of lochia and serum cytokines levels before and after treatment were compared. Results The bleeding amount at 0.5 h, 2 h, 24 h after administration in the observation group were (76.82±15.40) mL, (112.34±18.73) mL and (196.70±20.60) mL, respectively, which were significantly lower than those in the control group[(147.38±17.65), (198.49±19.37) mL, (283.74±21.56) mL](t=17.565, 18.643, 17.020, all P<0.05). The duration of contractions[(3.83±1.40) h] and the extent of uterine decline[(6.25±0.93) cm] in the observation group were significantly better than those in the control group[(1.92±0.59) h, (4.65±0.66) cm], and the hemostasis time[(21.29±3.60)min] and duration of lochia[(17.19±4.67)d] in the observation group were significantly shorter than those in the control group[(42.28±3.85) min, (24.28±6.11) d](t=23.220, 7.331, 8.181, 5.376, all P<0.05). After treatment, the levels of nitrogen monoxide (NO)[(91.22±6.23) μmol/L], nitric oxide synthase (NOS)[(24.56±2.46) μmol/L], brain natriuretic peptide (BNP)[(46.81±5.10)ng/L] in the observation group were lower than those in the control group[(98.63±7.51) μmol/L, (30.92±3.95) μmol/L, (90.35±4.66)ng/L], and the differences were statistically significant (t=4.428, 7.969, 36.750, all P<0.05). Conclusion The effect of ergometrine maleate injection combined with posterior pituitary injection on postpartum hemorrhage is effective, which can effectively control the amount of bleeding, shorten the time of hemostasis and promote postpartum recovery. Key words: Postpartum hemorrhage; Ergonovine; Vasotocin; Drug therapy, combination; Nitric oxide; Nitric oxide synthase; Natriuretic peptide, brain

Myocardial bridging is an independent predictor of positive spasm provocation testing by intracoronary ergonovine injections: a retrospective observational study

The relationship between myocardial bridging (MB) and coronary spasms during spasm provocation testing (SPT) remains unclear. We aimed to investigate whether MB was correlated with the SPT by ergonovine (ER) injections in a retrospective observational study. Of the 3340 patients who underwent a first coronary angiography, 166 underwent SPT using ER injections and were divided into 2 groups: MB(+) (n = 23), and MB(−) (n = 143). MB was defined as an angiographic reduction in the diameter of the coronary artery during systole. The patients who had severe organic stenosis in the left anterior descending coronary artery were excluded. The MB(+) group more frequently had diabetes mellitus and chronic kidney disease, and a thicker interventricular septum thickness. The rate of SPT-positivity was higher in the MB(+) group than MB(−) group (56.5% vs. 22.4%, P = 0.001). A multivariate regression analysis showed that the presence of MB was independently associated with SPT-positivity (odds ratio 5.587, 95% confidence interval 2.061–15.149, P = 0.001). In conclusion, coronary spasms during provocation tests with ER independently correlated with the MB. MB may predict coronary spasms.

Ergot Alkaloid Effects on Bovine Sperm Motility &amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt;

Cattle in some parts of the world graze pastures that consist of tall fescue that may contain ergot alkaloid contamination. Those ergot alkaloids are associated with reduced reproductive rates in cattle. Our objective was to determine if ergot alkaloids [dihydroergotamine (DHET), ergonovine (EN), and ergotamine (ET)] directly affect bovine sperm characteristics. Spermatozoa were collected from mature Angus (n = 2) and Balancer (n = 4) bulls. Within bull, treatments were structured as a 3 × 5 factorial with three alkaloids (DHET, EN, and ET) and five concentrations of each alkaloid (0, 33, 66, 100, or 200 μM). Spermatozoa (25 × 106) were incubated (39˚C) in 1 mL of modified sperm medium. Sperm motility characteristics were evaluated using CASA (Hamiliton Thorne IVOS, Beverly, MA) at 0, 3, and 6 h after initial alkaloid exposure. Initial sperm motility was (69% ± 1.1%) and declined (P = 0.01) to (35% ± 2.6%) at 6 h. Sperm motility decreased (P < 0.05) with increasing concentrations of DHET and ET, but not EN. As concentration of ET or DHET increased all CASA sperm characteristics were altered. The interaction of alkaloid concentration and incubation length affected sperm velocity and head size; exposure to 200 μM of ET or DHET for six hours decreased (P < 0.05) both characteristics. Our results demonstrate that ergot alkaloids (ET and DHET) can directly alter bovine sperm motility and morphology, which adds to our understanding of how ergot alkaloids may hinder cattle reproductive rates.

Safety of intracoronary provocative testing for the diagnosis of coronary artery spasm

AimsSystematic review of literature to evaluate safety of intracoronary (i.c.) pharmacologic testing with acetylcholine (ACh), or ergonovine (ERGO), to induce coronary artery spasm. Methods and resultsReview of all relevant publications using MEDLINE and EMBASE databases yielded 10 publications, totalling 9,444 patients. Prevalence of provoked spasm varied from 2.3% to 54.7% of patients tested in the selected studies. The wide variability in prevalence was due to heterogeneity of study populations and provocation protocols. No deaths were reported. Overall occurrence of major (0.8%) and minor (4.7%) complications for i.c. pharmacologic testing was low. Compared to ERGO, ACh showed significantly higher rate of major (1.09% vs 0.15%; p<0.001) and minor complications (5.87% vs 2.36%; p<0.001). ConclusionProvocative testing with i.c. ACh or ERGO are safe and can facilitate the diagnosis of inducible coronary artery spasm during diagnostic coronary angiography. These tests should be part of the routine armamentarium of interventional cardiologists.

Relationships among ergot alkaloids, cytochrome P450 activity, and beef steer growth.

Determining a grazing animal's susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 μM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 days of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 days. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = −0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

Serious cardiac complications in coronary spasm provocation tests using acetylcholine or ergonovine: analysis of 21 512 patients from the diagnosis procedure combination database in Japan.

Previous studies on complications with coronary spasm provocation tests were based on small sample sizes or were limited to high-volume centers. The risk of provocation tests using acetylcholine (ACH) or ergonovine (ER) remains to be fully examined by a large-scale multicenter study. ACH provocation tests are associated with a higher rate of serious cardiac complications than ER tests. Using the Diagnosis Procedure Combination database in Japan, we identified patients aged ≥20 years who underwent a pharmacological provocation test during coronary angiography. We assessed the composite outcome of cardiac complications requiring urgent procedures (defibrillation, chest compression, intra-aortic balloon pumping, or extracorporeal membrane oxygenation) or death on the day of the provocation test, and compared the outcome between ACH and ER tests. Of 21 512 eligible patients in 602 hospitals, 10 628 (49.4%) underwent an ACH test and 10 884 (50.6%) underwent an ER test. The composite outcome occurred in 141 (0.7%) patients. The ACH group was significantly more likely to have the composite outcome than the ER group (0.9% vs 0.4%, P < 0.001). The propensity-score analyses showed consistent results (propensity score-matched, 0.9% vs 0.4%, P = 0.003; inverse probability-weighted, 0.8% vs 0.4%, P < 0.001). In a multivariable logistic regression analysis, ACH tests were significantly associated with a higher rate of the composite outcome than ER tests (odds ratio: 1.75, 95% confidence interval: 1.13-2.69, P = 0.011). This retrospective cohort study suggested that ACH tests were associated with a higher rate of cardiac complications than ER tests.

Complications of Pharmacological Spasm Provocation Tests

Background: We investigated the complications of spasm provocation tests, including acetylcholine (ACh) tests, ergonovine (ER) tests and adding ACh after ER tests, retrospectively. Methods: We performed 1546 ACh tests and 1114 ER tests during 23 years, including 240 adding ACh after ER tests. ACh (RCA: 20/50/(80) g, LCA: 20/50/100/(200) g) was injected incrementally over 20 seconds, whereas ER (RCA: 40 g, LCA: 64 g) was administered over 2-4 minutes. In addition, we administered adding intracoronary injection of ACh (RCA: 50/80 g, LCA: 100/200 g) after ER tests. Serious major complications were defined as ventricular fibrillation, sustained ventricular tachycardia, shock, severe hypotension (< 60 mmHg), cardiac arrest and cardiac tamponade. Results: Serious major and major complications were higher in ACh tests than ER tests (1.8% vs. 0.4%, p<0.01), whereas serious major complications were not different between ACh and ER tests (0.9% vs. 0.4%, ns). No serious major or major complications were observed in adding ACh after ER tests. Paroxysmal atrial fibrillation was not different between ACh tests and adding ACh after ER tests (16.7% vs. 12.5%, ns). Necessity of nitrates to relive provoked spasms prior to carrying other site tests were significantly higher in ER tests (6.8%) than ACh tests (2.0%) and adding ACh after ER test (2.5%). No death or irreversible complications were recognized in all three spasm provocation tests. Conclusions: Serious major complications were not different between the two agents, whereas serious major and major complications were significantly higher in ACh tests than ER tests. Although pharmacological spasm provocation tests including adding ACh after ER tests were reliable and relatively safer methods, we should perform these tests positively in the cardiac catheterization laboratory.

Safety and optimal protocol of provocation test for diagnosis of multivessel coronary spasm.

We examined the safety of acetylcholine (ACh) and ergonovine (ER) tests retrospectively and investigated the optimal protocol of provocation test for diagnosis of multivessel coronary spasm. We performed 1546 ACh tests and 1114 ER tests during 23 years. ACh was injected in incremental doses of 20/50/80 μg into the right coronary artery (RCA) and of 20/50/100/200 μg into the left coronary artery (LCA) over 20 s. ER was administered in total doses of 40 μg into the RCA and of 64 μg into the LCA over 2-4 min. When a coronary spasm was induced and did not resolve spontaneously within 3 min after the completion of ACh/ER injection, or when hemodynamic instability due to coronary spasms occurred, 2.5-5.0 mg of nitrate was administered into the responsible vessel. To relive provoked spasm, it is necessary to administer nitrate in 31 cases by ACh and in 76 cases by ER (2.0 vs. 6.8 %, p < 0.01) before another vessel attempts. Multivessel spasms were often observed in LCA testing than in RCA testing on both agents [ACh: 78.6 % (11/14) vs. 11.8 % (2/17), p < 0.001, ER: 37.8 % (14/37) vs. 20.5 % (8/39), ns]. Even after the administration of nitrates, positive coronary spasm was obtained in 21.1 % by ACh and 52.9 % by ER tests on another coronary artery. No irreversible complications were recognized on both tests. We should firstly perform spasm provocation tests in the LCA and we may be able to diagnose another vessel spasm by performing the complete spasm provocation tests after the administration of nitrates to relieve provoked spasm in the first attempt.

Dietary exposure to ergot alkaloids decreases contractility of bovine mesenteric vasculature1,2

Ergot alkaloids are hypothesized to cause vasoconstriction in the midgut, and prior exposure may affect the vasoactivity of these compounds. The objectives of this study were to profile vasoactivity of ergot alkaloids in bovine mesenteric artery (MA) and vein (MV) and determine if previous exposure to endophyte-infected tall fescue seed affected vasoactivity of ergocryptine (ERP), ergotamine (ERT), ergocristine (ERS), ergocornine (ERO), ergonovine (ERN), lysergic acid (LSA), ergovaline-containing tall fescue seed extract (EXT), and 5-hydroxytryptamine (5HT; serotonin). Ruminally cannulated Angus steers (n = 12; BW = 547 ± 31 kg) were paired by weight and randomly assigned to 6 blocks. Steers were ruminally dosed daily with 1 kg of either endophyte-infected (E+; 4.45 mg ergovaline/kg DM) or endophyte-free (E-; 0 mg ergovaline/kg DM) tall fescue seed for 21 d before slaughter. Branches of MA and MV supporting the cranial portion of the ileum were collected after slaughter on d 22, placed in a modified Krebs-Henseleit buffer on ice, cleaned, sectioned, and mounted in a multimyograph chamber. Contractile response was normalized to a maximum KCl response. Inner diameter (P = 0.04) and outer diameter (P = 0.02) of MA were smaller for E+ steers than E- steers. Maximum contractile responses to 120 mM KCl were not different between seed treatments in MA (P = 0.33; E-: 2.67 ± 0.43 g; E+: 3.33 ± 0.43 g) or MV (P = 0.26; E-: 2.01 ± 0.18 g; E+: 1.81 ± 0.18 g). Steers receiving E+ had a smaller (P < 0.01) MA contractile response than E- steers to ERP, ERT, ERS, ERO, ERN, EXT, and 5HT. Steers receiving E+ had a smaller (P < 0.05) MV contractile response than E- steers to ERP, ERT, ERS, ERN, EXT, and 5HT. Lysergic acid failed to induce a contractile response in MA and MV. The contractile response in MA and MV of E- steers produced by 5HT was very large. The EXT was the most potent (P < 0.05) agonist in MV and MA of E+ steers. These data showed that ergot alkaloids were vasoactive in the bovine midgut, and steers exposed to E+ had diminished contractility to some ergot alkaloids in small intestinal vasculature. The findings of this study suggest that dietary exposure to ergot alkaloids has the potential to alter nutrient absorption from the midgut by decreasing blood flow to and from the midgut due to vasoconstriction.

Liver cytochrome P450 system as affected by endophyte-infected tall fescue seed extracts and ergot alkaloids

Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids. This study was designed to investigate the effects of E+ seed extract and selected ergot alkaloids on the detoxification pathway by cytochrome P450 (CYP3A4) enzyme system. Tests were performed using the P450- Glo CYP3A4 enzyme activity kit (Promega, WI), according to the manufacturer’s manual. Luminescence was measured using a single tube TD20/20 luminometer. Endophyte infected tall fescue seed was extracted with 50/50 methanol/25 mM ammonium carbonate, cleaned and concentrated on Strata-X reversed phase column (Phenomenex). The extracts were evaluated on an HPLC, and then tested using a serial dilution method. Commercially available ergonovine (EN), ergocorine (ER), bromocryptine (BC) and ergocryptine (EC) were tested individually using 0 to 44 nM concentrations. Seed extract of E+ produced a significant (P P < 0.05) dose dependent manner with EC being most potent, followed by ER, BC, and then EN (70%, 40%, 30% and 10% at 44 nM concentration). The similarity of the inhibition curves of seed extract to that of the commercially available ergot alkaloids suggests a related mode of action and that the use of such ergot alkaloids and CYP3A4 assay is a good model to study the toxicity of tall fescue. Furthermore, it provides the foundation to identify the individual toxic components of purified endophyte infected tall fescue extract.

Mass spectral characterization of ergot alkaloids by electrospray ionization, hydrogen/deuterium exchange, and multiple stage mass spectrometry: usefulness of precursor ion scan experiments

Six ergot alkaloids belonging to the lysergic acid derivatives (ergonovine (EGN) and methysergide hydrogen maleinate (MHM)) and peptide-type derivatives (ergocristine (EGR), ergotamine (EGT), ergocornine (EGC) and alpha-ergokryptine (EGK)) were studied by positive electrospray tandem mass spectrometry. The fragmentation mechanisms of these compounds were studied by collision-induced dissociation (CID) using triple quadrupole and ion trap mass spectrometers, and the nature of the major product ions further confirmed by hydrogen/deuterium (H/D) exchange experiments. A common abundant product ion at m/z 223 was characteristic of the two classes of ergot alkaloids. Therefore, a precursor ion scan of m/z 223 that triggers information data acquisition (IDA) in combination with CID experiments was used to identify other potential ergot alkaloids. Using this approach, it was possible to confirm the presence of ergosine, another peptide-type ergot alkaloid, in a rye flour extract at trace levels.

331 DIRECT EFFECTS OF ERGOT ALKALOIDS ON BOVINE SPERM MOTILITY IN VITRO

Ergot alkaloids have been associated with decreased livestock reproductive rates. Concentration of alkaloids in the reproductive tract after consumption of toxic forage is unknown. In addition, the direct effects of alkaloids on bovine spermatozoa have not been determined. We investigated the direct and interactive effects of 3 ergot alkaloids (ergotamine, dihydroergotamine, and ergonovine) on motility of frozen–thawed bovine spermatozoa. Thawed spermatozoa from 3 bulls were pooled and washed using a Percoll density gradient. Sperm motility was visually estimated by counting at least 100 spermatozoa in each triplicate well (&amp;gt; 300 per treatment per replicate). The cell counting was conducted using phase contrast (400�) on an inverted bright field microscope. Spermatozoa were considered motile if they exhibited free progressive forward or other movement and were not attached to the well surface. Motile spermatozoa were exposed to alkaloids ranging in concentration from 0 to 100 �M. Assays were conducted in modified sperm-TL (mSPTL) medium at 39�C in moist air without CO2 for 6 to 12 h. The results showed that both ergotamine (ET) and dihydroergotamine (DHET) inhibited (P &amp;lt; 0.05) sperm motility at concentrations greater than 50 �M and 33.3 �M, respectively. Ergonovine (EN) did not inhibit sperm motility at the test concentrations. Inhibitory effects of alkaloids on sperm motility were concentration-dependent for ET and DHET incubations and time-dependent for DHET incubations. Sperm motility also was inhibited by an interaction (P &amp;lt; 0.05) between ET and DHET at concentrations of 16.7 �M or above. The medium pH affected the toxic effects of both ET and DHET, whereas the medium osmolarity affected only the toxic effect of ET on relative sperm motility (P &amp;lt; 0.05). Medium osmolarity of 358 mOsm and/or pH higher than 7.1 exacerbated the toxic effects of the alkaloids. These results demonstrate that ergot alkaloids can directly interact with spermatozoa and impair sperm motility. Herbivores consuming toxic tall fescue are exposed to a cocktail of ergot alkaloids. Alkaloid interactive effects coupled with altered cell chemistry, due to increased respiration rates and frequent urination, on spermatozoa may indicate the mechanism by which reproduction is impaired in animals consuming toxic forage.

Clinical impact of selective spasm provocation tests: comparisons between acetylcholine and ergonovine in 1508 examinations

There are few reports regarding the concordance of coronary arterial response between acetylcholine (ACh) and ergonovine (ER) spasm provocation tests. We attempted to perform selective spasm provocation tests to examine the incidence of provoked spasm in patients who had undergone first coronary angiography as much as possible and we compared the coronary arterial response and clinical usefulness between selective intracoronary injection of ACh and intracoronary administration of ER. We performed 1508 selective spasm provocation tests, consisting of 873 ACh tests and 635 ER tests, from 1991 to 2002. We examined the frequency of provoked spasms of each agent retrospectively. ACh was injected in incremental doses of 20, 50 and 80 microg into the right coronary artery and 20, 50 and 100 microg into the left coronary artery. ER was administered as 10 microg/min over 4 min for a maximal dose of 40 microg in the right coronary artery and as 16 microg/min over 4 min for a total dose of 64 microg in the left coronary artery. Coronary spasm was defined as transient >99% luminal narrowing. Intracoronary ACh provoked spasms in 36.0% of patients and intracoronary ER induced spasms in 29.8% of patients. In patients with ischemic heart disease, the incidence of provoked spasms was not different between ACh tests (50.9%) and ER tests (43.8%). In contrast, the frequency of provoked spasms with ACh tests was significantly higher than that with ER tests (11.0% compared with 6.4%, P<0.05) in patients without ischemic heart disease. Moreover, ACh provoked more spasms in patients without fixed stenosis than ER (36.2% compared with 25.5%, P<0.01) and multiple spasms were frequently observed when performing ACh tests (40.0% compared with 27.0%, P<0.01). Major complications were observed in 1.4% of patients with ACh tests and in 0.2% of patients with ER tests. The need for intracoronary administration of isosorbide dinitrate to relieve coronary spasms during ER testing before performing another coronary artery test was more frequently observed in ACh tests (5.04% compared with 1.49%, P<0.01). However, no serious irreversible complications, such as death or acute myocardial infarction, were observed in this study. There was a significant difference in sex, history of smoking and hyperlipidemia between patients with and without spasms for both tests, whereas no difference in age or hypertension was observed in either test. Thus, both selective ACh and ER tests were useful as spasm provocation tests.

Antagonistic effects of simultaneous exposure of ergot alkaloids on kidney adenosine triphosphatase system.

Much of the research on fescue toxicosis has concentrated on evaluating animal response to grazing endophyte-infected (E+) versus endophyte-free tall fescue or the effects of single toxins such as ergonovine (EN), ergovaline (EV), or ergotamine (ET) on animal performance. Such approaches have eliminated the opportunity to test the possible additive, synergistic, or antagonistic interactions of one or more ergot alkaloids with the other ergot alkaloids found in E+ tall fescue. This study was conducted to determine the effects of simultaneous exposure of pairs of EN, EV, and ET on the kidney adenosine triphosphatase (ATPase) system in vitro. Tests were performed using three separate rat kidney homogenates and were repeated four times at concentrations of 0, 75, and 200 microM. Individually, EN, EV, and ET induced dose-dependent inhibitions of kidney Na(+)/K(+) ATPase, with EN being most potent, followed by purified EV, and then by ET. The ergot alkaloids inhibited Mg(2+) ATPase to a lesser degree than Na(+)/K(+) ATPase, with EN again being the most potent toxin. Simultaneous exposure to any combination of the ergot alkaloid pairs tested (EV + ET, EV + EN, and ET + EN) resulted in significant interactions (P < 0.05), indicating antagonistic effects on the inhibition of Na(+)/K(+) ATPase and Mg(2+) ATPase for most concentration combinations. These interactions suggest that in studies of the effects of any ergot alkaloid on animal performance, effects of other ergot alkaloids may also be present. Effects may not be additive, as was the case in this study, and the presence of one toxin may enhance or hinder the effectiveness of others.

Reproductive hormonal responses to ergotamine and ergonovine in cows during the luteal phase of the estrous cycle.

We conducted research to evaluate whether ergot alkaloids associated with endophyte-infected tall fescue could alter plasma concentrations of hormones important to reproductive function in cows. Six primiparous, estrous-cycling Holstein cows nursing calves received single i.v. injections of ergotamine tartrate, ergonovine maleate, or saline vehicle in a simple cross-over design. Each cow received one compound per estrous cycle, d 15 or 16 after estrus, and all compounds over three estrous cycles. Blood samples were collected at 20-min intervals for 40 min before and 240 min after treatments to assess plasma concentrations of prolactin, LH, FSH, and the response by PGF2alpha metabolite 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Ambient temperature and respiratory rates were measured hourly. Ambient temperature averaged 26 degrees C during data collection. Treatment x time was a significant source of variation for respiration rate and plasma concentrations of every hormone evaluated except FSH. Ergotamine elevated (P < .01) respiration rates 2 to 4 h after treatment. Ergotamine and ergonovine reduced (P < .001) plasma concentrations of prolactin for 2 to 4 h after treatment compared to concentrations before treatment. Plasma concentrations of LH were lowered (P < .01) by ergonovine during the 3rd h after treatment and by ergotamine during the 4th h after treatment. Most cows exhibited secretory PGFM responses after ergotamine and ergonovine treatments compared with an absence of a PGFM response in all cows after saline (P < or = .01). Results indicated that ergot alkaloids implicated as causative agents for fescue toxicosis can alter plasma concentrations of reproductive hormones during the late luteal phase of the estrous cycle in cows.