Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile

Crystallization-inhibiting proteins can explain longer nucleation times associated with bile from gallstone-free subjects as compared with bile from patients with cholesterol gallstones. We partially characterized and examined the crystallization inhibitory potency of a newly purified 15 kd human biliary protein. Gallbladder bile was passed through an anti-apolipoprotein A-I (apo A-I) immunoaffinity column to extract lipid-associaied proteins. The bound fraction was separated by 30 kd ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) was performed under nonreducing and reducing conditions. Cholesterol crystallization activity was tested in a photometric cholesterol crystal growth assay. Isoelectric focusing was performed by using a standard gel, The purified 15 kd protein was subjected to N-terminal amino acid sequencing, Although the whole apo A-I-bound fraction contained a variety of proteins and lipids, its 30 kd filtrate yielded a nearly pure 15 kd protein with only minor contamination from apo A-I. Amino acid sequencing showed that the protein was unique. Enzymatic deglycosylation revealed no evidence for glycosylation. At a protein concentration of 10 mu g/ml, crystallization time was delayed as compared with control and apo A-I, and final crystal mass was reduced to 75% of control, Its isoelectric point was 6.1 without isoforms, Under nonreducing conditions, the protein formed a 30 kd dimer and a 60 kd tetramer. We conclude that this protein is a novel potent biliary crystallization inhibitor protein

BILIARY HAPTOGLOBIN, A POTENT PROMOTER OF CHOLESTEROL CRYSTALLIZATION AT PHYSIOLOGICAL CONCENTRATIONS

Background/Aims: Several proteins present in human bile have been reported to promote cholesterol crystallization and thus are potentially important in the formation of cholesterol crystals as the initial stage in gallstone pathogenesis. To be physiologically relevant, such proteins must either be present in high concentration in bile or have a potent promoting activity. The current study explored several of the more abundant but unexamined biliary proteins based upon their also having sufficiently high serum concentrations that antibodies were available for both their isolation and quantitation. Methods: Protein purification was accomplished by immunoaffinity chromatography of bile followed by delipidation. Con A affinity chromatography of bile was used to obtain the bound fraction, a portion of which was delipidated. Crystallization-promoting activity of both the purified proteins and Con A-bound glycoprotein fractions (CABG) was measured by a photometric crystal growth assay. A competitive antibody-capture ELISA assay was developed to measure concentrations of alpha(1)-antitrypsin, transferrin, and haptoglobin in native bile. Results: At their relevant physiological concentrations, biliary haptoglobin (15 mu g/ml) had a crystallization-promoting activity twice that of the biliary IgM (75 mu g/ml) used as a reference standard (P < 0.05). Biliary transferrin (20 mu g/ml) had only modest promoting activity (P < 0.05). Biliary alpha(1)-antitrypsin (50 mu g/ml), by contrast, showed no promoting activity. Delipidation of the CABG fraction decreased its promoting activity by 75%. Biliary haptoglobin accounts for about 30% of delipidated total CABG-promoting activity. Conclusions: Biliary haptoglobin at its physiological concentration has a highly potent crystallization-promoting activity and thus becomes a candidate for major attention in understanding gallstone pathogenesis. Biliary lipids associated with CABG account for a major portion of the cholesterol-crystallization-promoting activity of this fraction

Complications during pharmacological stress echocardiography: a video-case series

BACKGROUND: Stress echocardiography is a cost-effective tool for the modern noninvasive diagnosis of coronary artery disease. Several physical and pharmacological stresses are used in combination with echocardiographic imaging, usually exercise, dobutamine and dipyridamole. The safety of a stress is (or should be) a major determinant in the choice of testing. Although large scale single center experiences and multicenter trial information are available for both dobutamine and dipyridamole stress echo testing, complications or side effects still can occur even in the most experienced laboratories with the most skilled operators. CASE PRESENTATION: We decided to present a case collection of severe complications during pharmacological stress echo testing, including a ventricular tachycardia, cardiogenic shock, transient ischemic attack, torsade de pointe, fatal ventricular fibrillation, and free wall rupture. CONCLUSION: We believe that, in this field, every past complication described is a future complication avoided; what happens in your lab is more true of what you read in journals; and Good Clinical Practice is not "not having complications", but to describe the complications you had

Feasibility, safety and tolerability of accelerated dobutamine stress echocardiography

A continuous infusion of a single high dose of dobutamine has been, recently, suggested as a simple and effective protocol of stress echocardiography. The present study assesses the feasibility, safety, and tolerability of an accelerated dobutamine stress protocol performed in patients with suspected or known coronary artery disease. Two hundred sixty five consecutive patients underwent accelerated dobutamine stress echocardiography: the dobutamine was administered at a constant dose of 50 μg/kg/min for up to 10 minutes. The mean weight-adjusted cumulative dose of dobutamine used was 330 ± 105.24 μg/kg. Total duration of dobutamine infusion was 6.6 ± 2.1 min. Heart rate rose from 69.9 ± 12.1 to 123.1 ± 22.1 beats/min at peak with a concomitant change in systolic blood pressure (127.6 ± 18.1 vs. 167.6 ± 45.0 mmHg). Dobutamine administration produced a rapid increase in heart rate (9.4 ± 5.9 beats/min2). The side effects were similar to those described with the standard protocol; the most common were frequent premature ventricular complexes (21.5%), frequent premature atrial complexes (1.5%) and non sustained ventricular tachycardia (1.5%); among non cardiac symptoms the most frequent were nausea (3.4%), headache (1.1%) and symptomatic hypotension (1.1%). No major side effects were observed during the test. Our data demonstrate that a continous infusion of a single high dose of dobutamine is a safe and well tolerated method of performing stress echocardiography in patients with suspected or known coronary artery disease. This new protocol requires the administration of lower cumulative dobutamine dose than standard protocol and results in a significant reduction in test time

Evolution of Dobutamine Echocardiography Protocols and Indications: Safety and Side Effects in 3,011 Studies Over 5 Years

AbstractObjectives. This study sought to document the safety of dobutamine stress echocardiography as it has evolved at a single center and to define predictors of adverse events.Background. The indications and protocol for dobutamine stress testing have evolved over 5 years of clinical use, but the influence of these changes on the safety and side effects of the test is undefined.Methods. Over 5 years, 3,011 consecutive dobutamine stress studies were performed in 2,871 patients, using an incremental protocol from 5 to 40 μg/kg body weight per min in 3-min stages, followed by atropine or an additional stage with 50 μg/kg per min, if required. Clinical data were gathered prospectively, and hemodynamic and echocardiographic findings were recorded at each stage, including recovery. Dobutamine echocardiography was defined as positive for ischemia in the presence of new or worsening wall motion abnormalities; in the absence of ischemia, failure to attain 85% of age-predicted maximal heart rate was identified as a nondiagnostic result.Results. Studies were performed for risk assessment (70%) and symptom evaluation (30%); over the study period, there was an increment in the use of dobutamine echocardiography for preoperative evaluation. Most tests (n = 2,194 [73%]) were terminated due to attainment of peak dose with achievement of target heart rate (>85% maximal age-predicted heart rate); 455 patients (15%) failed to achieve >85% maximal predicted heart rate despite maximal doses of dobutamine and atropine. The protocol was stopped prematurely in 230 patients (7.6%) because of side effects, including ventricular (n = 27 [0.9%]) and supraventricular rhythm disorders (n = 22 [0.7%]), severe hypertension (n = 24 [0.8%]) and hypotension or left ventricular outflow tract obstruction (n = 112 [3.8%]). Noncardiac symptoms, such as headache, nausea or anxiety, caused early test termination in 45 patients (1.6%). The remaining tests were stopped because of severe chest pain (n = 106 [3.5%]) or severe ischemia by echocardiography (n = 26 [0.9%]). Serious complications occurred in nine patients, including sustained ventricular tachycardia in five, myocardial infarction in one and other conditions in three requiring hospital admission (sustained supraventricular tachycardia, hypotension, suspected myocardial infarction), but neither ventricular fibrillation nor death occurred. Independent predictors of serious complications could not be defined. Over 5 years, higher dose protocols and more frequent use of atropine have raised the number of diagnostic protocols from 59% to 80%, without increasing the incidence of major side effects.Conclusions. Despite the use of more aggressive protocols and alterations of the indications for testing to include sicker patients, major side effects are a rare complication of dobutamine echocardiography.(J Am Coll Cardiol 1997;29:1234–40