Determination of hydroxyalkyl derivatives of cobalamin (vitamin B12) using reversed phase high performance liquid chromatography with electrospray tandem mass spectrometry and ultraviolet diode array detection.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) and ultraviolet diode array detection (UV-DAD), coupled on-line to reversed phase high performance liquid chromatography (HPLC), was used for the characterization of hydroxyalkyl derivatives of cob(I)alamin. The reduced form of vitamin B12, cob(I)alamin, denoted a supernucleophile due to its high nucleophilic strength, has shown promise as an analytical tool in studies of electrophilically reactive compounds in vitro and in vivo. A method for analysis of DNA-phosphate adducts was developed earlier utilizing the supernucleophilicity of cob(I)alamin to transfer alkyl groups from the phosphotriester configuration in DNA, with the formation of a Co-substituted alkyl-cobalamin (alkyl-Cbl) complex. For the purpose of identification and quantification of alkyl-Cbls at high sensitivity, an MS/MS method has been developed with application to a number of 2-hydroxyalkyl-cobalamins (OHalkyl-Cbls). The precursor oxiranes were reacted with cob(I)alamin, followed by clean-up and mass spectrometric analysis of the resulting OHalkyl-Cbls. It was found that ionization was highly dependent on solvent composition. By using acetonitrile/water/trifluoroacetic acid (TFA) (eluent I), the base peak was the doubly protonated molecule [M + 2H](2+), whereas acetonitrile/water/1-methylpiperidine (eluent II) yielded the singly protonated molecule [M + H](+) as the base peak. Excellent separation was obtained with eluent II, with good separation between stereoisomers, thus enabling the characterization of these by means of UV spectra. Limits of quantitation for 2-hydroxypropyl-cobalamin (OHPr-Cbl) were 0.2 and 2 pg/microL (or 0.1 and 1 fmol/microL) using selected ion recording (SIR) with eluent I and II, respectively. The obtained detection level should be sufficient for analysis of alkyl-Cbls from a wide range of toxicological applications.

Adverse events with transvenous implantable cardioverter-defibrillators: a prospective multicenter study. European 7219 Jewel ICD investigators.

BACKGROUND: A newly developed classification system relates adverse events to the surgical procedure or the function of the implantable defibrillator. METHODS AND RESULTS: Adverse events were monitored during prospective clinical evaluation of the Medtronic model 7219 Jewel ICD and were classified according to the definitions of the ISO 14155 standard for device clinical trials into 3 groups: severe and mild device-related and severe non-device-related adverse events. In addition, events were related to the surgical procedure, treatment with the device, or cardiac function. Seven hundred seventy-eight patients were followed up for an average of 4.0 months after ICD implantation. In total, 356 adverse events were observed in 259 patients. At 1, 3, and 12 months after ICD implantation, 99%, 98%, and 97% of the patients, respectively, survived; 95%, 93%, and 92%, respectively, were free of surgical reintervention; and 79%, 68%, and 51%, respectively, were free of any adverse event. Twenty patients died: 6 deaths were related to the surgical procedure, 12 deaths were considered unrelated to ICD treatment, and 2 patients died of an unknown cause. Of 111 nonlethal severe adverse device effects, 47 required surgical intervention, 19 times for correction of a dislodged lead. Inappropriate delivery of therapy was observed 128 times in 111 patients, and the events were typically resolved by reprogramming or drug adjustment. Nine of these required rehospitalization. CONCLUSIONS: Approximately 50% of patients experience an adverse event within the first year after ICD implantation. The observed adverse event rate depends on the definitions and the prospective monitoring. The incidence of inappropriate therapy emphasizes the need for improved detection algorithms and for quality-of-life evaluations, especially when considering ICD treatment in high-risk but arrhythmia-free patients.

Free polymerized hemoglobin versus hydroxyethyl starch in resuscitation of hypovolemic dogs.

Polymerized bovine hemoglobin (PBH) was compared with hydroxyethyl starch (HES) in a hypovolemic shock model. Eighteen dogs were subjected to hemorrhage; systolic arterial blood pressure was maintained at 40 mm Hg for 30 min (mean blood pressure 37.8 +/- 4.7 [SD] mm Hg). Resuscitation was conducted by infusing their own shed blood (control group) or 6% HES (mol wt 200,000) in 0.9% NaCl (HES group) or PBH (PBH group), both in an equal amount to the shed blood. Directly after infusion, oxygen delivery and consumption returned to prehemorrhage levels in all three groups. In the HES group, the lowered arterial oxygen content was compensated by a 158% increase in cardiac output, in contrast to an increase of 31% and 9%, respectively, in the control and PBH groups. Early recovery from hypovolemic shock with regard to oxygen transport and delivery in the PBH group seemed to be comparable to the control group, without the increase in cardiac output seen with HES infusion.

Fluorochemical emulsion APE-LM substantially improves cardiac preservation.

We determined the efficacy of a novel fluorochemical emulsion for long-term hypothermic preservation of hearts. Rat hearts were preserved for 12 h at 12 degrees C with use of continuous low-pressure coronary perfusion with one of three oxygenated media (n = 6 hearts/groups): an "extracellular" crystalloid solution; APE-LM, a novel fluorochemical emulsion of perfluoroperhydrophenanthrene in egg yolk phospholipid; and FC-43, the Fluosol-43 (Oxypherol) fluorochemical emulsion of perfluorotributylamine in Pluronic F68. The emulsion media contained the same components as the crystalloid medium. All three media contained 0.5% albumin. An isolated working heart perfusion system was used to quantify the function of preserved hearts and controls (fresh hearts, n = 6). The APE-LM-preserved hearts were not significantly different from control hearts in contractile function, output, and energetics during a 4-h 37 degrees C reperfusion period. The control and APE-LM-preserved hearts had significantly better performance than crystalloid- and FC-43-preserved hearts. All preserved hearts gained fluid during preservation. The edema of APE-LM-preserved hearts, but not that of the other two preserved groups, was reversed during 37 degrees C reperfusion. These data provide the first evidence that a unique fluorochemical emulsion improves long-term preservation of cardiac tissue and produces significantly better recovery of cardiac function after preservation. This salutary effect was specifically associated with APE-LM emulsion and may result from its high O2 capacity, its biologically compatible emulsifier, and its superior physical properties, which include very small emulsion particle size (0.1-0.15 micron), low viscosity, and minimal toxicity.

31P-NMR study of cardiac preservation: St. Thomas' Hospital cardioplegic solution versus UW preservation solution.

Ex vivo cardiac preservation was evaluated by measuring the catabolism of high-energy phosphate (ATP and creatine phosphate, CrP) using 31P-NMR spectroscopy. After cardioplegic arrest St. Thomas' Hospital cardioplegic solution (group A), and University of Wisconsin (UW) preservation solution (group B) were tested. The hearts were mounted in the 4.7 T horizontal bore magnet of the NMR spectrometer and were continuously perfused with the test solution under 25 cm H2O pressure for 6 h at 10 degrees C. Peak heights of the beta-phosphate of ATP and CrP were measured and expressed as percentages of the initial value. For both group A and group B. ATP declined less rapidly during preservation than CrP. In group A, ATP remained constant for 60 min while CrP decreased from the onset of preservation. After 6 h of preservation 28.3% of ATP and 24.5% of CrP remained (group A). On the other hand, in group B, levels of both ATP and CrP remained much more stable: CrP did not decrease during the first 3 h of preservation, while ATP started to decrease after 5 h. At the end of preservation 76.1% of ATP and 71.5% of CrP were still present. We conclude that UW solution is superior to St. Thomas' Hospital solution for the preservation of high-energy phosphates during 6 h cardiac preservation with continuous hypothermic low-flow perfusion.

Catabolism of high energy phosphates during long-term cold storage of donor hearts: effects of extra- and intracellular fluid-type cardioplegic solutions and calcium channel blockers.

Dog hearts were harvested and stored cold (0.5 degree C) for 24-hours. Cardiac arrest was induced by means of low-sodium and calcium-free cardioplegic (n = 6) or hyperkalemic cardioplegic (n = 6) solution. Nifedipine (2 micrograms/gm estimated heart weight) was added to each cardioplegic solution in two additional groups (n = 6 each). High energy phosphates (creatine phosphate and adenosine triphosphate) and catabolites (adenosine diphosphate and monophosphate, adenosine, inosine, hypoxanthine, xanthine) were determined in the myocardium before and during 24 hours of cold storage. With use of the standard hyperkalemic cardioplegic solution, breakdown of high energy phosphates was less pronounced than after the use of a low sodium, calcium-free solution: after 24 hours of cold storage myocardial ATP content was 57% of control versus 32% (p less than 0.05). The addition of nifedipine to the hyperkalemic cardioplegic solution delayed ATP breakdown during the first hours of cold storage: at 5 hours of preservation the myocardial ATP level was significantly higher (p less than 0.05) than in hearts preserved without nifedipine. Addition of nifedipine to the low-sodium, calcium-free solution did not influence catabolism of high energy phosphates significantly. It is concluded that preservation of high energy phosphates during long-term cold storage of donor hearts can be best achieved by simultaneous myocardial metabolic blockade at two specific sites: at the "fast" sodium-potassium channels by hyperkalemic depolarization and at the "slow" channels by means of calcium channel blockers.

A comparison of selected health-related data from surveys of a general population and a sporting population.

This paper presents a comparison of health-related data from two surveys of British adults. Respondents from one survey formed a representative sample of the British population, whilst respondents from the other were characterised by their participation in sport. Statistical analysis revealed that sports participants had significantly lower body mass index values, lower blood pressures and lower resting pulse rates (P less than 0.05). In addition, sports participants possessed better self-perceived health and perceived themselves overall to be more active than members of the general population. When controls were introduced for gender and age, many of these differences remained, particularly among females. It is concluded that despite the cross-sectional nature of these data, evidence is provided that supports the health-promoting case for sport.

Differences in high-energy phosphate catabolism between the rat and the dog in a heart preservation model.

Concentrations of ATP and creatine phosphate were measured in rat and dog hearts preserved either by cold storage (procedure A) or by continuous hypothermic perfusion (procedure B). In procedure A (3 dogs, 4 rats) the hearts were normothermically excised without cardioplegia and were stored in 0.9% NaCl at 0.5 degrees C; in procedure B (6 dogs, 21 rats) hypothermic cardioplegic arrest was performed, and then the hearts were retrogradely perfused through the aorta for 24 hours with use of an oxygenated Bretschneider cardioplegic solution at 2 degrees to 4 degrees C. Whole rat hearts were frozen using Wollenberger clamps at desired times during the preservation period; transmural needle biopsy specimens were sampled from dog hearts. In control nonpreserved hearts, the ATP and creatine phosphate were as follows (mean +/- SD): 26.7 +/- 4.1 and 27.1 +/- 10.3 mumol/gm dry weight, respectively (dog hearts), and 23.1 +/- 2.1 and 34.2 +/- 12.1 mumol/gm dry weight, respectively (rat hearts). With procedure A, ATP decreased by 36% in dog hearts and by 64% in rat hearts during the first hour of storage. By 24 hours, only 6% of the ATP remained in the dog hearts and 1% in the rat hearts. Creatine phosphate decreased by 85% (dog hearts) and by 93% (rat hearts) during the first hour of storage. The ATP and creatine phosphate values observed in rat hearts after 1 hour of procedure A preservation were significantly lower than in dog hearts (p less than 0.05). With procedure B, cardioplegic arrest by itself did not alter high-energy phosphate concentrations in dog or rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioprotective effects of carnitine in extensive aortocoronary bypass grafting: a double-blind, randomized, placebo-controlled clinical trial.

The cardioprotective effects of carnitine were tested in patients undergoing multiple aortocoronary bypass grafting. Intermittent aortic cross-clamping at 28 degrees C was used. Mean total cross-clamping time was 30 +/- 11 min. Patients were randomized into three groups: a control group receiving placebo (group 1), a group pretreated with 3 g carnitine intravenously before cardiopulmonary bypass (CPB) (group 2), and a group pretreated with 6 g carnitine intravenously (group 3). The markers of myocardial ischemia included levels of adenosine triphosphate, its catabolites, and creatine phosphate in transmural left ventricular biopsy specimens taken at the beginning and end of CPB, as well as hemodynamic recovery during weaning from CPB and for the next 24 h. The intravenous infusion of carnitine (3 or 6 g) had no hemodynamic effect. At the end of CPB myocardial tissue levels of adenosine triphosphate and creatine phosphate did not differ significantly among the groups (P greater than 0.05). Recovery of cardiac function during weaning from CPB and for the following 24 h was similar in all three groups (P greater than 0.05). It is concluded that pretreatment with carnitine neither facilitates weaning from cardiopulmonary bypass in patients undergoing aortocoronary bypass surgery nor favorably affects hemodynamic function during the next 24 h.

Long-term preservation of donor hearts: the effect of intra- and extracellular-type of cardioplegic solutions on myocardial high energy phosphate content.

The influence of cardioplegic arrest (single or multidose cardioplegia) and subsequent long-term cold storage on myocardial high energy phosphate content was studied in 29 dogs divided into 6 groups of experiments. Three cardioplegic solutions were tested: Bretschneider HTK (intracellular-type solution), St. Thomas' Hospital and N.I.H. solutions (both extracellular-type solutions). In group I, II and III single dose cardioplegic arrest with respectively St. Thomas' Hospital, Bretschneider HTK and N.I.H. solutions was carried out and excised hearts were stored at 0.5 degrees C for 24 hours. In group IV-Bretschneider HTK and in group V-N.I.H. solutions were used for cardioplegic arrest and intermittent perfusion of the cooled hearts at 4, 8 and 12 hours of storage (multidose cardioplegia). In group VI, after cardioplegic arrest with Bretschneider HTK solution, different temperatures of storage (0.5 degrees C, 12 degrees C and 18 degrees C) were studied. Myocardial content of ATP and creatine phosphate was evaluated by means of bioluminescence techniques from serial left ventricular biopsies taken prior to aortic cross-clamping and after 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours. In group I ATP was significantly lower than in groups II and III after 6 h (p less than 0.005). After 24 hours of storage ATP-levels were significantly higher (p less than 0.05) after multidose Bretschneider HTK cardioplegia or N.I.H. cardioplegia than after single dose N.I.H. or St. Thomas' cardioplegia. There was no significant difference in ATP content between multidose Bretschneider and multidose N.I.H. cardioplegia.(ABSTRACT TRUNCATED AT 250 WORDS)

Successful orthotopic heart transplantation in dogs after 24 hours of continuous perfusion with Bretschneider HTK cardioplegic solution.

In 12 dogs the hearts after excision were perfused for 24 hours with Bretschneider HTK cardioplegic solution. Six of these hearts were used only to assess myocardial HEP and ultrastructure during 24 hours of conservation. In the next six dogs orthotopic heart transplantation was performed to evaluate functional outcome after prolonged preservation. After 24 hours of continuous perfusion of the donor heart the ATP level was completely comparable with control, preischemic value. Also ultrastructure of the myocytes was perfectly preserved. All transplanted hearts recovered completely upon reperfusion without a need of inotropic support. Good functional outcome after transplantation was correlated with about 70% of myocardial HEP content and intact ultrastructure of the myocytes. We concluded that continuous perfusion with Bretschneider HTK cardioplegic solution makes successful heart transplantation possible after 24 hours of preservation.

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay.

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.

Optimal storage temperature and benefit of hypothermic cardioplegic arrest for long-term preservation of donor hearts: a study in the dog.

Currently, for practical clinical purposes, the preservation of donor hearts is limited to about 4 h. Transplantation must be finished within this period to assure complete functional recovery upon reperfusion. From the clinical setting it is well known that hypothermia results in a better myocardial preservation during ischemia. During ischemia, rapid catabolism of high-energy phosphates (e.g., ATP and creatine phosphate) occurs. The purpose of this study was to investigate the influence of temperature during a 24-h preservation period on the rate of catabolism of ATP and on the rate of accumulation of breakdown products (ADP, AMP, adenosine, inosine, hypoxanthine, and xanthine). For this purpose, hearts were excised and stored for 24 h at 0.5 degrees, 12 degrees, or 18 degrees C. In addition, the effect of initial cardioplegic arrest was compared with simple normothermic excision of the heart followed by 24 h in cold storage. It was found that the higher the storage temperature, the higher the rate of catabolism of high-energy phosphates and, hence, after 24 h, the lower the final ATP level and the higher the level of breakdown products, mainly nucleosides. It was also found that the initial cardioplegic arrest strongly benefits the preservation of high-energy phosphates as a result of the ATP-sparing effect.

Energy state of the myocardium during long-term cold storage and subsequent reperfusion.

Long-term preservation of dog hearts was performed over 24 h using Bretschneider-HTK cardioplegia and cold storage. Preservation was assessed in terms of conservation of myocardial tissue levels of high-energy phosphates (HEP) and functional outcome after cardiac transplantation. Serial left ventricular biopsies were taken and analysed for ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine and creatine phosphate. Myocardial structure was studied by electron microscopical examination of a similar biopsy specimen. Cardiac performance was measured before and after cardiac transplantation. Several techniques of cardioplegic arrest were studied: single dose cardioplegia, multidose cardioplegia and continuous perfusion with the cardioplegic solution. In all groups, the hearts were stored at 0.5 degree C for 24 h. In the group of single dose Bretschneider-HTK cardioplegia, myocardial ATP content after 24 h of cold storage was only 25% of control. The total sum of nucleotides at that time interval was however 65% of the control value. Reperfusion of these hearts using a support dog (whole blood reperfusion) did not result in any recovery of ATP. Creatinine phosphate however showed an overshoot. Accumulated nucleosides were washed out. The hearts showed electrical activity but were severely arrhythmic. Contractility was poor. In the group of multidose Bretschneider-HTK cardioplegia, HEP preservation was better than after single dose cardioplegia. ATP content was about 50% of control. The total sum of nucleotides was 85% of control. Ultrastructural assessment of the myocytes revealed only slight ischaemic damage to the mitochondria. Reperfusion on cardiopulmonary bypass after cardiac transplantation did not show any restoration of ATP, but a steady catabolism of HEP. The nucleosides adenosine and inosine were not washed out upon reperfusion. After cardiac transplantation, none of these hearts could be weaned from cardiopulmonary bypass due to irreversible low cardiac output. Histological examination demonstrated irreversible myocardial tissue damage. In the group of continuous cold Bretschneider cardioplegia, HEP content was completely preserved throughout the 24 h of perfusion. Ultrastructure of the myocytes was normal. Reperfusion of the transplanted hearts showed a mild breakdown of ATP to 70% of control values accompanied by a slight accumulation of nucleosides. Haemodynamic recovery however was perfect and none of the hearts needed positive inotropic support. Myocytes after reperfusion had a normal subcellular appearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Relation between coronary artery stenosis and myocardial purine metabolism, histology and regional function in humans.

In 54 patients undergoing elective or emergency aortocoronary bypass grafting, angiographic and electrocardiographic changes were studied. Five patients with unstable angina and five patients with evolving myocardial infarction were included. High energy phosphate metabolism and the histologic appearance of the myocardium were analyzed in transmural biopsy specimens acquired at the time of surgery. In patients without anterior infarction on the electrocardiogram, severe stenosis of the left anterior descending coronary artery resulted in a reduction of anterior wall motion that was associated with a partial depletion of the adenylate pool. Mitochondrial function, however, remained intact: the adenosine diphosphate/adenosine triphosphate ratio, the energy charge and the creatine phosphate/adenosine triphosphate ratio were in the normal range. Histologic assessment demonstrated viable myocardium with a high incidence of atrophic cells. In evolving myocardial infarction, 170 minutes of acute coronary artery obstruction resulted in anterior wall akinesia associated with a decrease of the sum of the adenylates to 52% and of creatine phosphate to 16% of their normal value (p less than 0.05). The nucleosides accumulated; their major fraction (91%) was inosine. The adenosine diphosphate/adenosine triphosphate ratio increased from 0.14 +/- 0.04 to 0.49 +/- 0.20 (p less than 0.01) and the energy charge decreased from 0.924 +/- 0.021 to 0.660 +/- 0.169 (p less than 0.01). Ultrastructure examination revealed irreversible cell damage in at least the subendocardial layer. These results suggest that the energetic base of reduced contractility due to severe coronary artery stenosis is different from that in acute coronary obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of areal densities of blood vessel wall components in histological sections by means of image analysis.

A technique is described which allows quantification of specifically stained, intermingled vessel wall components in paraffin-embedded tissue. The dissociation of elastic, muscular and connective tissue and the determination of their areal densities in the superior vena cava and in the ascending aorta of the dog, was performed by automated image analysis. The percentages of elastic+connective tissue, determined by this technique, correlated significantly with the data derived from biochemical measurements. It was further found that the structure of the vena cava wall was a function of the distance from the right atrium.

Geometrical variations of the canine superior vena cava: relationship between diameter, segment length and transmural venous pressure.

The dimensional variations of the superior vena cava throughout the cardiac cycle were investigated by means of angiographic and ultrasonic techniques. Results obtained by both methods were largely in agreement. In frontal direction the diameter curve resembled the transmural pressure variations, the sagittal diameter curve was not related to the venous pressure variations during certain phases of the cardiac cycle and compressional and/or stretching forces exerted by respectively the distending aorta and/or pulmonary artery and the contracting heart may have been responsible. Also segment length variations occurred: during atrial contraction, ventricular systole and early diastole the vein elongated, indicating the varying longitudinal traction forces, possibly exerted by the rotational movement of the contracting heart. Assuming elliptical symmetry the vena caval cross-section could be reconstructed, and the cross-sectional area, perimeter and segmental volume could be calculated throughout the cardiac cycle. Despite the high venous distensibility, no extreme collapse of the vein was observed during the cardiac cycle at low transmural pressures.

Correlation between mechanical properties and wall composition of the canine superior vena cava.

The mechanical properties (modulus of elasticity and stress-relaxation) of different venous segments of the canine superior vena cava were determined as well as the composition of the vessel wall by means of physical, biochemical and histological methods. It was found that the wall of the vena cava was structurally and mechanically a function of the metric position with respect to the right heart: the modulus of elasticity increased, the stress-relaxation decreased, the concentration of hydroxyproline, collagen and elastin increased and the amount of muscle fibres decreased with increasing distal distance from the right heart. A significant linear correlation coefficient was observed between the modulus of elasticity and the structural wall components. The data presented show the axial heterogeneity and the dependency of the mechanical properties upon the venous vessel wall composition.