Mass spectrometry-guided refinement of chemical energy buffers.

Biocatalytic reactions often require supplying chemical energy and phosphate groups in the form of adenosine triphosphate (ATP). Auxiliary enzymes can be used to convert a reaction by-product-adenosine diphosphate (ADP)-back to ATP. By employing real-time mass spectrometry (RTMS), one can gain an insight into inter-conversions of reactants in multi-enzyme reaction systems and optimize the reaction conditions. In this study, temporal traces of ions corresponding to adenosine monophosphate (AMP), ADP and ATP provided vital information that could be used to adjust activities of the 'buffering enzymes'. Using the RTMS results as a feedback, we also characterized a bienzymatic energy buffer that enables the recovery of ATP in the cases where it is directly hydrolysed to AMP in the main enzymatic reaction. The significance of careful selection of enzyme activities-guided by RTMS-is exemplified in the synthesis of glucose-6-phosphate by hexokinase in the presence of a buffering enzyme, pyruvate kinase. Relative activities of the two enzymes, present in the reaction mixture, influence biosynthetic reaction yields. This observation supports the conclusion that optimization of chemical energy recycling procedures is critical for the biosynthetic reaction economy.

Fusion of microlitre water-in-oil droplets for simple, fast and green chemical assays.

A simple format for microscale chemical assays is proposed. It does not require the use of test tubes, microchips or microtiter plates. Microlitre-range (ca. 0.7-5.0 µL) aqueous droplets are generated by a commercial micropipette in a non-polar matrix inside a Petri dish. When two droplets are pipetted nearby, they spontaneously coalesce within seconds, priming a chemical reaction. Detection of the reaction product is accomplished by colorimetry, spectrophotometry, or fluorimetry using simple light-emitting diode (LED) arrays as the sources of monochromatic light, while chemiluminescence detection of the analytes present in single droplets is conducted in the dark. A smartphone camera is used as the detector. The limits of detection obtained for the developed in-droplet assays are estimated to be: 1.4 nmol (potassium permanganate by colorimetry), 1.4 pmol (fluorescein by fluorimetry), and 580 fmol (sodium hypochlorite by chemiluminescence detection). The format has successfully been used to monitor the progress of chemical and biochemical reactions over time with sub-second resolution. A semi-quantitative analysis of ascorbic acid using Tillman's reagent is presented. A few tens of individual droplets can be scanned in parallel. Rapid switching of the LED light sources with different wavelengths enables a spectral analysis of multiple droplets. Very little solid waste is produced. The assay matrix is readily recycled, thus the volume of liquid waste produced each time is also very small (typically, 1-10 µL per analysis). Various water-immiscible translucent liquids can be used as the reaction matrix: including silicone oil, 1-octanol as well as soybean cooking oil.

Mass spectrometric method incorporating enzymatic amplification for attomole-level analysis of target metabolites in biological samples.

We report a sensitive method for selective detection of target metabolites from the central metabolic pathway by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), in which the MS signal is enhanced by up to three orders of magnitude in the course of in situ enzymatic amplification.

Brachial approach directional coronary atherectomy of a left coronary artery saphenous vein bypass graft.

Directional coronary atherectomy (DCA) of a saphenous vein bypass graft to the left coronary artery was performed percutaneously from the brachial artery approach using a 7F endomyocardial biopsy sheath. Initial positioning was accomplished with a left bypass graft catheter inserted in the sheath. This technique permits use of smaller catheters than usual for DCA in patients in whom larger guides cannot be used.

Randomized comparison of over-the-wire and fixed-wire balloon devices for coronary angioplasty.

One hundred and fifteen consecutive patients undergoing coronary angioplasty (PTCA) were randomized to low profile over-the-wire (OTW, e.g., Mini, Sulp II) or fixed-wire (FW, e.g., Ace, Orion, Probe) PTCA balloon wire devices. Overall success, ability to begin and finish with the same balloon device, fluoroscopy time, physician time, and amount of contrast media used were prospectively assessed. Overall final success rate was excellent regardless of which type of system was used first (OTW 92%; FW 96%). In 6 patients no wire would cross, in another, the stenosis would not dilate despite high pressure ( greater than 18 atm). When an OTW device was used first, it was initially successful in 69% of patients and was changed with success to a second OTW balloon in 4 (cumulative 76%). An OTW balloon was changed to FW in 11 patients (success 9 patients, 82%). This was because of inability to cross with balloon of the OTW system after a steerable wire had crossed in 9 of the patients. When a FW device was used first, it was initially successful in 89%. A change to another FW device led to success in 4 additional patients (cumulative 96%). In no patient was a FW changed to an OTW device. Thus, a FW device was more likely to be successful (either 54 of 56 patients initially randomized to FW, or 63 of 67 total patient number in whom a FW device was used) than an OTW device (45 of 59 patients) p less than .05.(ABSTRACT TRUNCATED AT 250 WORDS)

Atenolol and chlorthalidone therapy for hypertension: a double-blind comparison.

In a randomized, double-blind, parallel-group study of 31 patients with mild to moderate hypertension, we compared a placebo regimen with a regimen of atenolol and chlorthalidone (Tenoretic). The study, which lasted seven weeks, began with a single-blind two-week placebo lead-in period, followed by a four-week double-blind treatment phase, and concluded with a one-week single-blind placebo washout period. Of 24 patients included in the analysis of efficacy, seven received one Tenoretic 50 tablet per day (atenolol, 50 mg; chlorthalidone, 25 mg), nine received one Tenoretic 100 tablet per day (atenolol, 100 mg; chlorthalidone, 25 mg), and eight received placebo. Supine systolic/diastolic blood pressure (mean +/- SD) decreased from 154 +/- 15.2/102 +/- 4.6 mm Hg during the baseline period to 128 +/- 8.5/85 +/- 4.0 mm Hg during treatment in the group receiving Tenoretic 100, from 153 +/- 12.6/104 +/- 5.4 mm Hg to 137 +/- 4.5/91 +/- 4.4 mm Hg in the group receiving Tenoretic 50, and from 150 +/- 11.9/101 +/- 1.6 mm Hg to 145 +/- 11.6/93 +/- 5.1 mm Hg in the group receiving placebo. Reductions in systolic and diastolic blood pressures in the active treatment groups were significantly greater than the pressure reductions in the group receiving placebo (P less than .05 to .1). The combination of atenolol and chlorthalidone was well tolerated, and in no case was treatment discontinued because of side effects. This study showed that one tablet per day of either Tenoretic 50 or Tenoretic 100 is effective and well tolerated in the treatment of mild to moderate hypertension.

Intracoronary thrombolysis in acute myocardial infarction: clinical course following successful myocardial reperfusion.

We reviewed the clinical course of 73 patients who had attempted intracoronary thrombolysis, with emphasis on follow-up. Fifty-nine patients (81%) had coronary reflow sufficient to control pain and injury current: 52 received thrombolysis alone and seven had thrombolysis combined with acute coronary angioplasty. Recurrent ischemic events in hospital were frequent and occurred in 17 patients (29%). These included silent reocclusion (four patients), recurrent angina (eight patients), and recurrent infarction in the same myocardial zone (five patients). Late ischemic events occurred in 11 patients (19%) and included silent reocclusion (two patients) and angina (nine patients). Although acute coronary angioplasty resulted in a high rate of successful myocardial reperfusion, long-term vessel patency was infrequent. The results of coronary bypass surgery, performed in hospital for severe residual coronary stenosis and angina and later for recurrent angina, were uniformly good. At follow-up of 6 to 36 months (mean 18.5 +/- 8.1), total mortality was five patients (8%). Only 16 reperfused patients (27%) were alive and well without recurrent ischemia or interventions. We conclude that reopening an acutely occluded coronary artery by thrombolysis and/or angioplasty can be performed in the majority of patients but must be regarded as initial therapy in view of the high incidence of recurrent ischemic events. Reperfused patients with stable myocardial blood supply post infarction have low long-term mortality.

Reperfusion arrhythmia: a marker of restoration of antegrade flow during intracoronary thrombolysis for acute myocardial infarction.

We studied the effects of coronary recanalization on arrhythmogenesis in patients undergoing intracoronary thrombolysis during the early hours of myocardial infarction. Catheterization, ventriculography, coronary angiography, and intracoronary streptokinase infusion were performed in 22 patients. Twenty-one of 22 had thrombotic total occlusion of the infarct-related transient thrombolysis with reocclusion by the end of the procedure. In 12 of these 17 patients, restoration of antegrade coronary flow was accompanied by transient arrhythmia. In these 12 patients coronary angiography within seconds of onset of arrhythmia showed vessel patency in a previously totally occluded coronary artery. Two additional patients developed arrhythmias during streptokinase infusion but after reperfusion had already been established. Accelerated idioventricular rhythm was most often noted. Sinus bradycardia and atrioventricular block with hypotension occurred during restoration of flow in arteries supplying the inferoposterior left ventricle. These arrhythmias may be useful noninvasive markers of successful reperfusion during thrombolytic therapy in acute myocardial infarction.

Combination therapy for evolving myocardial infarction: intracoronary thrombolysis and percutaneous transluminal angioplasty.

Nonsurgical coronary reperfusion for evolving myocardial infarction is a promising new technique for the salvage of jeopardized myocardium. Successful reperfusion can be established by intracoronary infusion of streptokinase in approximately 75 percent of patients within the first 6 hours of transmural infarction [1.2]. Following recanalization, most patients are left with high grade fixed coronary stenoses which are potential sites for recurrent thrombus formation. Since the underlying site for coronary thrombosis is still present, reocclusion may occur. Indeed, early experience suggests that recurrence of thrombosis is not uncommon [3.4]. Therapy for evolving myocardial infarction should, in some patients, involve not only thrombolysis, but also an attack on the fixed coronary lesion. We describe a patient wit evolving myocardial infarction who was treated successfully with combination therapy consisting of intracoronary streptokinase followed by percutaneous transluminal coronary angioplasty [5].