Visualization of lipid droplet composition by direct organelle mass spectrometry.

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.

Safety and efficacy of the argatroban therapy during the early post-cardiac surgery period.

Heparin-induced thrombocytopenia (HIT) is associated with a high incidence of vein graft occlusion after cardiac surgery. When HIT is suspected during the post-operative period, current guideline recommends a direct thrombin inhibitor such as argatroban to be started immediately. The aim of this retrospective study was to evaluate the safety and efficacy of argatroban in the early period after cardiac surgery. All patients who received argatroban within 72 h after cardiac surgery from September 2005 to June 2009 from a single center were included. Patient demographics, pre-operative relevant history, intra-operative events and post-operative data were collected and analyzed. The primary endpoints were bleeding, thrombotic complication during or after argatroban administration, and in-hospital mortality. The study population comprised 31 patients administered argatroban within 72 h after cardiac surgery. Argatroban was started a mean of 1.7 days after surgery (median dose, 0.66 µg/kg/min; median duration, 5.9 days). Twenty patients (64.5%) experienced bleeding; episode driven entirely by the need for blood transfusion. No new thromboembolic complication occurred during or after argatroban infusion. One patient died from aspiration pneumonia. Compared to those without bleeding complications, patients who bled had longer operation times and increased use of intra-aortic balloon pump. However, argatroban therapy including the starting time, median dose, infusion duration, and activated partial thromboplastin times showed no difference between the two groups. In cardiac surgery patients with clinical suspicion of HIT, early postoperative use of argatroban seems well-tolerated and associated with a low risk of thrombotic events.

Reduced argatroban doses after coronary artery bypass graft surgery.

BACKGROUND: The Food and Drug Administration-approved argatroban dose for heparin-induced thrombocytopenia (HIT) is 2 microg/kg/min (0.5 microg/kg/min in hepatic impairment), adjusted to achieve activated partial thromboplastin time (aPTT) 1.5-3 times baseline. Recent data suggest that reduced doses are required after cardiovascular surgery. OBJECTIVE: To characterize dosing requirements, aPTTs, factors affecting dosage, and clinical outcomes in patients administered argatroban after coronary artery bypass graft (CABG) surgery. METHODS: Charts of 39 patients who underwent CABG surgery and were administered argatroban postoperatively for laboratory-confirmed HIT (n = 25), antibody-negative suspected HIT (n = 10), or previous HIT requiring anticoagulation (n = 4) were retrospectively reviewed. Patient characteristics, argatroban dosing information, aPTTs (target range 45-90 sec), and outcomes were summarized. Regression analyses explored potential effectors of dosage. RESULTS: Patient features, argatroban dosing patterns, and aPTTs were similar among groups. Many patients had laboratory evidence of some hepatic and/or renal dysfunction (median [range] bilirubin 1.0 [0.3-8.0] mg/dL, creatinine clearance 47 [18-287] mL/min). Overall, median argatroban doses were 0.5 microg/kg/min initially and 0.6 microg/kg/min during therapy (median duration 5.3 days). After argatroban initiation, aPTTs were greater than 90 seconds at first assessment in 4 patients (3 with abnormal hepatic function test results) initially administered 0.5, 1, 2, and 2 microg/kg/min, respectively. Within approximately 16 hours of therapy, 33 (85%) patients achieved consecutive therapeutic aPTTs. No association was detected between mean dose during therapy and preoperative ejection fraction, routine hepatic or renal function test results (other than blood urea nitrogen [BUN]), or surgery type. A clinically insignificant association existed between dose and BUN: there was an approximately 0.15 microg/kg/min dose decrease for each 10 mg/dL BUN increase. One patient developed thrombosis, 1 underwent finger amputation, 7 died (5 after argatroban cessation), and 4 had significant bleeding. CONCLUSIONS: These findings suggest that reduced initial argatroban doses (eg, 0.5 microg/kg/min), adjusted to achieve therapeutic aPTTs, provide rapid, adequate anticoagulation in postoperative CABG patients with presumed or previous HIT. Prospective study of reduced initial dosing in this setting is warranted.

Randomization in clinical trials of titrated therapies: unintended consequences of using fixed treatment protocols.

OBJECTIVE: Clinical trial designs that randomize patients to fixed treatment regimens may disrupt preexisting relationships between illness severity and level of therapy. The practice misalignments created by such designs may have unintended effects on trial results and safety. METHODS: To illustrate this problem, the Transfusion Requirements in Critical Care (TRICC) trial and the Acute Respiratory Distress Syndrome Network low tidal volume (ARMA) trial were analyzed. RESULTS: Publications before TRICC indicated that clinicians used higher transfusion thresholds in patients with ischemic heart disease compared with younger, healthier patients (p = .001). The trial, however, randomized patients (n = 838) to liberal (10 g/dL hemoglobin) or restrictive (7 g/dL) transfusion thresholds. Thirty-day mortality was different and opposite in the liberal compared with the restrictive arm depending on presence (21 vs. 26%) or absence (25 vs. 16%) of ischemic heart disease (p = .03). At baseline in ARMA, consistent with prior publications, physicians set ventilator volumes lower in patients with high airway pressures and poor compliance (8.4-10.6 mL/kg interquartile range) than patients with less severe abnormalities (9.6-12 mL/kg) (p = .0001). In the trial, however, patients (n = 861) were randomized to low (6 mL/kg) or high (12 mL/kg) tidal volumes. In patients with low compliance (<0.6 mL/kg), 28-day mortality was higher when tidal volumes were raised rather than lowered (42 vs. 29%), but this effect was reversed in patients with higher compliance (21 vs. 37%; p = .003). CONCLUSIONS: In TRICC and ARMA, randomization to fixed treatment regimens disrupted preexisting relationships between illness severity and therapy level. This created noncomparable subgroups in both study arms that received care different and opposite from titrated care, that is, practice misalignments. These subgroups reduced the interpretability and safety of each trial. Characterizing current practice, incorporating current practice controls, and using alternative trial designs to minimize practice misalignments should improve trial safety and interpretability.

Prevalence of heparin/platelet factor 4 antibodies before and after cardiac surgery.

BACKGROUND: The clinical significance of heparin/platelet factor 4 (PF4) antibodies in subjects undergoing cardiac surgery has not been systematically studied. We prospectively investigated whether the presence of heparin/PF4 antibodies would predict clinical thrombosis in this population. METHODS: In 299 patients scheduled for cardiac surgery between October 2003 and March 2005, the heparin/PF4 antibodies and platelet count were measured immediately prior to, and 5 days after, surgery. The patients were followed up at 30 days for thrombotic complications. RESULTS: The prevalence of the heparin/PF4 antibodies was 4.3% (13 of 299) prior to surgery and increased more than fivefold to 22.4% (62 of 277) postoperatively (p < 0.0001). Thromboembolic events occurred in 8.8% of patients with negative antibody and in 6.3% of patients with positive antibody (p = 0.77). Of the 62 patients with positive heparin/PF4 antibodies postoperatively, 22 (35.5%) were treated with a nonheparin anticoagulant. There was a trend toward higher rates of thromboembolic events in subjects who were thrombocytopenic compared with those who were not (17.1% and 6.7%, respectively, p = 0.06), regardless of antibody status. Two out of 8 patients (25%) with both thrombocytopenia and a positive antibody (clinical heparin-induced thrombocytopenia [HIT]) suffered a thromboembolic event, compared with 17 of 222 (7.7%) without clinical HIT (p = 0.13). CONCLUSIONS: The high prevalence of antibodies to the heparin/PF4 complex after cardiac surgery and the low rate of thromboembolic complications in this population suggest that the antibody alone does not confer an increased risk of thrombotic complications. Monitoring for thrombocytopenia is recommended.