Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

NMR and Thermal Studies for the Characterization of Mass Transport and Phase Separation in Paracetamol/Copovidone Hot-Melt Extrusion Formulations.

The formulation of drug/polymer amorphous solid dispersions (ASDs) is one of the most successful strategies for improving the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). Hot-melt extrusion (HME) is one method for preparing ASDs that is growing in importance in the pharmaceutical industry, but there are still substantial gaps in our understanding regarding the dynamics of drug dissolution and dispersion in viscous polymers and the physical stability of the final formulations. Furthermore, computational models have been built to predict optimal processing conditions, but they are limited by the lack of experimental data for key mass transport parameters, such as the diffusion coefficient. The work presented here reports direct measurements of API diffusion in pharmaceutical polymer melts, using high-temperature pulsed-field gradient NMR. The diffusion coefficient of a model drug/polymer system (paracetamol/copovidone) was determined for different drug loadings and at temperatures relevant to the HME process. The mechanisms of the diffusion process are also explored with the Stokes-Einstein and Arrhenius models. The results show that diffusivity is linked exponentially to temperature. Furthermore, this study includes rheological characterization, differential scanning calorimetry (DSC), and 1H ssNMR T1 and T1ρ measurements to give additional insights into the physical state, phase separation, and API/polymer interactions in paracetamol/copovidone ASD formulations.

Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides.

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.

Asymmetric substitutions of 2-lithiated N-boc-piperidine and N-Boc-azepine by dynamic resolution.

Proton abstraction of N-tert-butoxycarbonyl-piperidine (N-Boc-piperidine) with sBuLi and TMEDA provides a racemic organolithium that can be resolved using a chiral ligand. The enantiomeric organolithiums can interconvert so that a dynamic resolution occurs. Two mechanisms for promoting enantioselectivity in the products are possible. Slow addition of an electrophile such as trimethylsilyl chloride allows dynamic resolution under kinetic control (DKR). This process occurs with high enantioselectivity and is successful by catalysis with substoichiometric chiral ligand (catalytic dynamic kinetic resolution). Alternatively, the two enantiomers of this organolithium can be resolved under thermodynamic control with good enantioselectivity (dynamic thermodynamic resolution, DTR). The best ligands found are based on chiral diamino-alkoxides. Using DTR, a variety of electrophiles can be used to provide an asymmetric synthesis of enantiomerically enriched 2-substituted piperidines, including (after Boc deprotection) the alkaloid (+)-beta-conhydrine. The chemistry was extended, albeit with lower yields, to the corresponding 2-substituted seven-membered azepine ring derivatives.

Asymmetric lithiation-substitution of amines involving rearrangement of borates.

Asymmetric lithiation of substituted benzylamines, N-Boc-pyrrolidine, or N-Boc-indoline using Beak's methodology was followed by electrophilic quench with trialkylboranes. The resulting borate intermediates rearrange with concomitant C-N bond breakage to give, after oxidation, chiral secondary alcohols with high enantioselectivity.

Dynamic resolution of N-Boc-2-lithiopiperidine.

Dynamic thermodynamic resolution of N-Boc-2-lithiopiperidine is possible using a chiral ligand; the two enantiomers of this organolithium can be resolved with selectivities of up to 85 : 15 from a selection of 26 chiral diamino-alkoxide ligands screened.

Dynamic kinetic and kinetic resolution of N-Boc-2-lithiopiperidine.

Asymmetric substitution of 2-lithiopiperidines can be achieved by dynamic resolution; the organolithium is formed as a racemic mixture by proton abstraction (or tin-lithium exchange) and is resolved in the presence of a chiral ligand.

Online monitoring of a photocatalytic reaction by real-time high resolution FlowNMR spectroscopy.

We demonstrate how FlowNMR spectroscopy can readily be applied to investigate photochemical reactions that require sustained input of light and air to yield mechanistic insight under realistic conditions. The Eosin Y mediated photo-oxidation of N-allylbenzylamine is shown to produce imines as primary reaction products from which undesired aldehydes form after longer reaction times. Facile variation of reaction conditions during the reaction in flow allows for probe experiments that give information about the mode of action of the photocatalyst.

Prosthesis-patient mismatch affects survival after aortic valve replacement.

BACKGROUND: Surgeons traditionally avoid the use of "small" aortic prostheses because of the potential for residual left ventricular outflow tract obstruction and persistent transvalvular gradients. This study examines the ratio between prosthetic valve size and the body surface area (BSA) of patients undergoing aortic valve replacement (AVR). We sought to determine the effect of potential "prosthesis-patient" mismatch on long-term survival. METHODS AND RESULTS: Follow-up was conducted on 2981 patients who underwent AVR with stented bioprostheses between 1976 and 1996. To account for differences between manufacturers' labeled valve sizes, we calculated the ratio between the prosthetic valve effective orifice area (EOA) and the patient's BSA (recorded for 2154 patients). The lowest decile in this cohort had a calculated EOA/BSA of <0.75 cm(2)/m(2) (Small group, n=227) compared with the control group (n=1927), in whom the EOA/BSA ratio was >0.75 cm(2)/m(2). Operative mortality was higher in the Small group (8% versus 5%, P:=0.03). Actuarial survival at 12 years was 50+/-5% in the Small group compared with 49+/-2% in the control group (P:=0.27). However, freedom from valve-related mortality was significantly lower in the Small group (75+/-5% versus 84+/-2%, P:=0.004). Cox regression analysis determined age and NYHA functional class to be the multivariate predictors of overall mortality, whereas advanced age and EOA/BSA <0.75 cm(2)/m(2) were found to be the predictors of valve-related mortality. CONCLUSIONS: Prosthesis-patient mismatch results in significantly higher early and late mortality after bioprosthetic AVR. We recommend careful selection of stented bioprostheses to ensure an adequate ratio of EOA to BSA. An EOA/BSA ratio of >0.75 cm(2)/m(2) may avoid residual left ventricular outflow tract obstruction and persistent transvalvular gradients. Careful prosthesis-patient matching will improve both early and late survival after AVR.

Aortic valve repair for adult congenital heart disease: A 22-year experience.

BACKGROUND: Aortic valve-preserving procedures have resulted in excellent outcomes in selected patients, particularly those with normal aortic valve leaflets and dilated aortic roots. However, several congenital heart lesions are associated with abnormal aortic valve leaflets. The long-term results of aortic valve repair for these lesions are not well defined. METHODS AND RESULTS: We reviewed the clinical records of 54 adult (age >18 years) patients who underwent repair of congenital abnormalities of the aortic valve between 1976 and September 1999. Follow-up data were available on 52 (96%) patients (mean 50+/-67 months, range 1 to 266). Patients underwent repair at a mean age of 34+/-14 years with associated diagnoses of subaortic stenosis (n=10), ventricular septal defect with prolapsing aortic valve (n=17), bicuspid aortic valve (n=23), sinus of Valsalva aneurysm (n=10), and bacterial endocarditis (n=2). There was 1 operative death (1.9%) and 3 late deaths. Survival at 5 and 10 years was 98+/-2% and 74+/-12%, respectively. Freedom from reoperation was 74+/-9% and 51+/-15% at 5 and 10 years, respectively. The presence of a ventricular septal defect predicted failure of valve repair (59% versus 22%, P:=0.01). A bicuspid aortic valve, subaortic stenosis, or the requirement for mitral valve surgery did not affect outcomes. CONCLUSIONS: Aortic valve repair in adult patients with congenital heart disease can be performed with minimal morbidity and mortality rates. The medium-term results of repair are acceptable, regardless of valvular or associated pathology. However, only 31 patients (57%) demonstrated long-term competence of the aortic valve, suggesting that most adult patients with congenital aortic valve disease will eventually require aortic valve replacement.

Hemodilution during cardiopulmonary bypass is an independent risk factor for acute renal failure in adult cardiac surgery.

BACKGROUND: This observational study sought to determine whether the degree of hemodilution during cardiopulmonary bypass is independently related to perioperative acute renal failure necessitating dialysis support. METHODS: Data were prospectively collected on consecutive patients undergoing cardiac operations with cardiopulmonary bypass from 1999 to 2003 at a tertiary care hospital. The independent relationship was assessed between the degree of hemodilution during cardiopulmonary bypass, as measured by nadir hematocrit concentration, and acute renal failure necessitating dialysis support. Multivariate logistic regression was used to control for variables known to be associated with perioperative renal failure and anemia. RESULTS: Of the 9080 patients included in the analysis, 1.5% (n = 134) had acute renal failure necessitating dialysis support. There was an independent, nonlinear relationship between nadir hematocrit concentration during cardiopulmonary bypass and acute renal failure necessitating dialysis support. Moderate hemodilution (nadir hematocrit concentration, 21%-25%) was associated with the lowest risk of acute renal failure necessitating dialysis support; the risk increased as nadir hematocrit concentration deviated from this range in either direction (P = .005). Compared with moderate hemodilution, the adjusted odds ratio for acute renal failure necessitating dialysis support with severe hemodilution (nadir hematocrit concentration <21%) was 2.34 (95% confidence interval, 1.47-3.71), and for mild hemodilution (nadir hematocrit concentration >25%) it was 1.88 (95% confidence interval, 1.02-3.46). CONCLUSIONS: Given that there is an independent association between the degree of hemodilution during cardiopulmonary bypass and perioperative acute renal failure necessitating dialysis support, patient outcomes may be improved if the nadir hematocrit concentration during cardiopulmonary bypass is kept within the identified optimal range. Randomized clinical trials, however, are needed to determine whether this is a cause-effect relationship or simply an association.

Myocardial infarction determined by technetium-99m pyrophosphate single-photon tomography complicating elective coronary artery bypass grafting for angina pectoris.

The incidence of acute myocardial infarction (AMI) complicating coronary artery bypass grafting (CABG) has previously been based on concordance of electrocardiographic, enzymatic and scintigraphic criteria. Technetium-99m pyrophosphate (Tc-PPi) single-photon emission computed tomography now enables detection of AMI with high sensitivity and specificity. Using this technique, perioperative AMI was detected in 12 of 58 patients (21%) undergoing successful elective CABG for stable angina pectoris. Stepwise multivariate logistic regression analysis was performed to compare the predictive value of preoperative (New York Heart Association class, left ventricular ejection fraction and use of beta blockers) and intraoperative (number of grafts constructed, use of internal mammary anastomoses, use of sequential saphenous vein grafts, smallest grafted distal vessel lumen caliber and aortic cross-clamp time) variables. Preoperative New York Association class (p = 0.04) and smallest grafted distal vessel lumen caliber (p = 0.03) were significant multivariate predictors of perioperative AMI. Only 1 perioperative patient with AMI (and 1 pyrophosphate-negative patient) developed new Q waves. Serum creatine kinase-MB was higher in patients with AMI by repeated measures analysis of variance (p = 0.0003). Five AMIs occurred in myocardial segments revascularized using sequential saphenous vein grafts, and 7 in segments perfused by significantly stenosed epicardial vessels with distal lumen diameter and perfusion territory considered too small to warrant CABG. At 6-month follow-up, the mean left ventricular ejection fraction increased from 0.61 to 0.65 in Tc-PPI-negative patients (p = 0.01), but not in perioperative patients with AMI.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of entrapped intracardiac air with intraoperative echocardiography.

Embolization of entrapped intracardiac air represents a significant risk to the patient undergoing open heart surgery. To date, there have been no menas available to ensure that the heart is free of air prior to restoration of the circulation. To assess whether M mode echocardiography can accurately detect intracardiac air, we studied 10 dogs during cardiopulmonary bypass. Randomly, air was or was not injected into the left ventricular cavity of the fibrillating heart. Intracardiac air could be recognized by the presence of a stippled granular pattern, or a loss of the discrete linear echoes or decreased far field echoes, or any combination of these three. In all, 131 random observations were made. When 1.0 cc of air was injected, sensitivity and specificity were both 100 percent, but when 0.2 cc was injected, sensitivity and specificity decreased to 86 and 58 percent, respectively. thus, M mode echocardiography appears to provide a sensitive and specific tool for detecting intracardiac air.

Left ventricular rupture after mitral valve replacement: endocardial repair with pericardial patch.

A patient with type I left ventricular rupture after mitral valve replacement was successfully treated by suturing a pericardial patch to the healthy endocardium around the tear.

An aortic valve-sparing operation for patients with aortic incompetence and aneurysm of the ascending aorta.

A number of patients who require an operation for complications of annuloaortic ectasia, such as aortic incompetence or aneurysm of the aortic root (or both), have normal aortic valve leaflets. We have treated these patients by excising the aneurysmal portion of the ascending aorta and sinuses of Valsalva but by leaving the aortic valve leaflets and some arterial wall attached to the left ventricular outflow tract. The aortic valve is reimplanted inside a collagen-impregnated tubular Dacron graft, similar to what is done for implantation of an aortic valve homograft. The coronary arteries are also reimplanted. This operation was performed in 10 patients. All patients had annuloaortic ectasia and five had the stigmata of Marfan syndrome. Four patients had acute aortic dissection. There were no operative deaths, but one patient required composite replacement of the aortic valve and ascending aorta because of persistent aortic incompetence. Postoperative Doppler echocardiography revealed normal aortic valve function in six patients and mild incompetence in three. The preliminary results of this new operation are encouraging. Further investigation is necessary to establish the best size, shape, and type of material that should be used to replace the aortic root while preserving the aortic valve.

Surgical treatment of postinfarction false aneurysm of the left ventricle.

Twelve patients underwent surgery for repair of false aneurysms of the left ventricle. The mean interval between the myocardial infarction and the diagnosis was 19 months (range 2 to 80 months). Congestive heart failure was the most common clinical presentation. Most patients had three-vessel coronary artery disease. The false aneurysm was posterior in 10 patients and anterior in two. Three patients with posterior aneurysm had severe mitral regurgitation. Repair was accomplished by resection of the false aneurysm and primary closure of the defect in four patients and by closure with a patch in eight. Nine patients also had coronary artery bypass. Mitral valve replacement was performed in three patients who had severe mitral regurgitation before the operation and in one patient who had severe mitral regurgitation after repair of the false aneurysm and could not be weaned from cardiopulmonary bypass. There were three operative deaths and one additional death after 2 months. All deaths occurred in patients who had mitral valve replacement. Eight patients survived the operation and remained well after a mean follow-up period of 62 months. Patients with false aneurysms of the left ventricle do well after surgical repair, except when concomitant mitral valve replacement is necessary.

Aortic valve replacement with stentless porcine aortic bioprosthesis.

Twenty-nine patients were entered in a clinical trial on aortic valve replacement with a stentless glutaraldehyde-fixed porcine aortic valve. This bioprosthesis is secured to the aortic root by the same technique used for aortic valve replacement with aortic valve homografts. The functional results obtained from this operation have been most satisfactory. To assess the hemodynamic benefit of eliminating the stent of a porcine aortic valve, we matched 22 patients with a stentless porcine bioprosthesis for age, sex, body surface area, valve lesion, and bioprosthesis size to 22 patients who had aortic valve replacement with a Hancock II bioprosthesis. Mean and peak systolic gradients across the aortic bioprosthesis and effective aortic valve areas were obtained by Doppler studies. Gradients across the stentless bioprosthesis were significantly lower than gradients across the Hancock II valve for every bioprosthesis size. Effective aortic valve areas of the stentless bioprosthesis were significantly larger than the valve areas of the Hancock II valve. Our data demonstrate that the hemodynamic characteristics of a glutaraldehyde-fixed porcine aortic bioprosthesis are greatly improved when the aortic root is used as a stent for the valve. This technique of implantation is expected to enhance the durability of the bioprosthesis, because the aortic root may dampen the mechanical stress to which the leaflets are subjected during the cardiac cycle.

Repair of the aortic valve in patients with aortic insufficiency and aortic root aneurysm.

Patients with aneurysms of the ascending aorta or aortic root frequently have aortic insufficiency despite normal aortic leaflets. The aortic valve dysfunction is caused by dilatation of the sinotubular junction, distortion or dilatation of the sinuses of Valsalva, annuloaortic ectasia, or a combination of these problems. In the case of annuloaortic ectasia, reconstruction of the aortic root is performed by reimplanting the aortic valve in a tubular Dacron graft (reimplantation). In the case of mild or no annuloaortic ectasia, reconstruction of the aortic root is performed by correcting the dilated sinotubular junction and replacement of the aortic sinuses if they are also dilated with an appropriately tailored Dacron graft (remodeling). From July 1989 to March 1994, 45 patients have had either reimplantation of the aortic valve (19 patients) or remodeling of the aortic root (26 patients). Fourteen patients had Marfan's syndrome, 11 had acute and five had chronic type A aortic dissection, and nine also had transverse arch aneurysm. There were two operative deaths, both in the remodeling group. One patient who had reimplantation needed composite replacement of the aortic valve and ascending aorta because of persistent aortic insufficiency after the repair. A young patient with Marfan's syndrome had progressive aortic valve dysfunction during a growth spurt and had aortic valve replacement 2 years after the initial operation. No other valve-related complication has occurred. The remaining 41 patients have only mild or no aortic insufficiency, and the repair remains stable from 1 to 58 months, mean 18 months. These two types of aortic valve reconstruction have provided excellent clinical results in carefully selected adult patients.

Mitral valve repair by replacement of chordae tendineae with polytetrafluoroethylene sutures.

Expanded polytetrafluoroethylene sutures have been used for replacement of diseased chordae tendineae during reconstructive procedures on the mitral valve in 43 patients. There were 28 men and 15 women whose mean age was 55 years, range 21 to 76. Three fourths of the patients were in New York Heart Association class III or IV. Replacement of primary chordae tendineae of the anterior leaflet was performed with 4-0 or 5-0 polytetrafluoroethylene sutures. A double-armed suture was passed twice through the fibrous portion of the papillary muscle head and tied down. Each arm of the suture was brought up to the free margin of the leaflet and passed through the area where the native chorda was attached. After the lengths of the two arms were adjusted, the ends were tied together on the ventricular side of the leaflet. Thirty patients had degenerative disease of the mitral valve; the incompetence was due to prolapse of the anterior leaflet in 14 patients and prolapse of the anterior and posterior leaflets in 16. Eleven patients had rheumatic mitral valve disease: four had stenosis, three had regurgitation, and four had mixed lesions. Two patients had ischemic mitral regurgitation caused by rupture of a papillary muscle head. There were no operative deaths. Patients have been followed up from 5 to 61 months, mean 13. Doppler echocardiographic studies were performed at regular intervals after the operation and revealed normal mitral valve function in most patients There were two failures that necessitated mitral valve replacement: one because of acute mitral regurgitation and the other because of hemolysis. There have been two late deaths, neither one valve related. Replacement of chordae tendineae with polytetrafluoroethylene sutures is simple and allows for reconstruction of the mitral valve in many patients who would otherwise require mitral valve replacement. Because our patients have been followed up for a limited time, the long-term results of this procedure remain unknown.

Postinfarction ventricular septal rupture: repair by endocardial patch with infarct exclusion.

A novel operative technique for postinfarction ventricular septal defect has been used in 44 consecutive patients. The operation consists of excluding rather than excising the infarcted septum and ventricular walls. This is accomplished by performance of a left ventriculotomy through the infarcted muscle and securing a glutaraldehyde-fixed bovine pericardium patch to the endocardium of the left ventricle all around the infarcted myocardium. The ventriculotomy is simply closed over the pericardial patch. There were 21 men and 23 women whose mean age was 69 +/- 7 years. Twenty-nine patients were in cardiogenic shock at the time of operation. All patients had Doppler echocardiography and coronary angiography before operation. All but two patients were operated on during the acute phase of the myocardial infarction. There were six operative deaths. Postoperative complications included renal failure in 10 patients and respiratory failure in 18. Severe right ventricular dysfunction was the only independent predictor of operative mortality. Patients have been followed up for a mean of 40 +/- 34 months. There have been six late deaths and three of these were because of cardiac problems. The actuarial survival at 6 years was 66% +/- 7%. Only one patient had a small residual ventricular septal defect. Late postoperative assessment of ventricular function by echocardiography revealed that most patients had normal or mild impairment of right ventricular function and mild or moderate impairment of left ventricular function. Repair of acute postinfarction ventricular septal defect by endocardial patch with infarct exclusion of the left ventricule probably avoids additional damage to the right ventricle, remodels the acutely infarcted left ventricle, and enhances survival.