The multicopper oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity.

The multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV-visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the multicopper oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site-directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 degrees C, a half-life of inactivation of approximately 6 h at 80 degrees C, and temperature values at the midpoint from 97 to 112 degrees C. McoP is an efficient metallo-oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min(-1), respectively, at 40 degrees C. It is noteworthy that McoP follows a ping-pong mechanism, with three-fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of multicopper oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.

The cis/trans isomerization of Cu(II)-bis-(glycinato) complex in solution: a computer aided multifrequency EPR and DFT/PCM calculation study.

In this paper the Cu(II)-bis-(glycinato) complex has been analysed in solution by applying a combined approach of multifrequency EPR and DFT/PCM calculations. The accuracy in the determination of magnetic parameters has been reached by the use of a unique simulation program (COSMOS) for the whole range of temperatures analysed and by the error analysis. A change in magnetic parameters was envisaged in the 243-253 K range of temperature, and was interpreted in terms of stabilization, near the freezing point of the solution, of one of the isomers of the complex. A DFT/PCM computational model was crucial in assigning, on the basis of the experimental superhyperfine interaction value, the isomer to the trans form.

Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering.

Iso-1-cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox-inspired strategy was implemented until an iso-1-cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15-fold higher total-turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein-based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox-inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.

Mechanism of versatile peroxidase inactivation by Ca(2+) depletion.

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.

A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multifrequency EPR and density functional theory study.

Versatile peroxidases are heme enzymes that combine catalytic properties of lignin peroxidases and manganese peroxidases, being able to oxidize Mn(2+) as well as phenolic and non-phenolic aromatic compounds in the absence of mediators. The catalytic process (initiated by hydrogen peroxide) is the same as in classical peroxidases, with the involvement of 2 oxidizing equivalents and the formation of the so-called Compound I. This latter state contains an oxoferryl center and an organic cation radical that can be located on either the porphyrin ring or a protein residue. In this study, a radical intermediate in the reaction of versatile peroxidase from the ligninolytic fungus Pleurotus eryngii with H(2)O(2) has been characterized by multifrequency (9.4 and 94 GHz) EPR and assigned to a tryptophan residue. Comparison of experimental data and density functional theory theoretical results strongly suggests the assignment to a tryptophan neutral radical, excluding the assignment to a tryptophan cation radical or a histidine radical. Based on the experimentally determined side chain orientation and comparison with a high resolution crystal structure, the tryptophan neutral radical can be assigned to Trp(164) as the site involved in long-range electron transfer for aromatic substrate oxidation.

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta.

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.

EPR characterization of mono(thiosemicarbazones) copper(II) complexes. Note II.

Copper(II) complexes with thiosemicarbazones have been shown to be more active in cell destruction, in the inhibition of DNA synthesis than the uncomplexed ligand. Several derivatives of thiosemicarbazones and their iron and copper complexes have been studied for their cytotoxicity and inhibiting activity against DNA synthesis. In the present work complexes formed in H2O-DMSO solution between copper(II) and the acetophenone thiosemicarbazone (ATSC) and the o-aminobenzaldehyde thiosemicarbazone (o-NH2TSC) have been studied. EPR studies have been performed at different pH values and metal-to-ligand ratios. The spectra have been recorded at both room (298 K) and low temperatures (120 K). A possible relationship between structure and activity is attempted on the basis of the EPR data.

EPR and O2.- scavenger activity: Cu(II)-peptide complexes as superoxide dismutase models.

Several copper(II) complexes with aminoacids and peptides are known to show superoxide dismutase (SOD)-like activity. EPR spectroscopy has proved to be a useful tool for studying the complex equilibria of the copper(II) ion and various ligands of biological importance in solution. In the present work, a variety of copper(II) complexes with di-, tri- and tetra-peptides containing only glycine residues (GG, GGG and GGGG) and others containing a histidyl residue in different positions (HGG, GHG, GGH and GGHG) have been investigated. EPR parameters obtained by extensive use of computer simulation of spectra lead to reliable spin Hamiltonian EPR parameters at both room temperature and in frozen solution. The molecular orbital coefficients computed from the anisotropic EPR data and the d-d electronic energies are used to characterize different arrangements of the complexes. Estimation of the scavenger activity of the complexes due to the particular environment created by the ligands around copper is discussed in the frame of the structure-activity relationship.

[Angiography compared to high resolution magnetic resonance and digital angiography in atherosclerosis of the iliac-femoral arteries]. / Studio comparativo tra angiografia con Risonanza Magnetica con alta risoluzione e angiografia digitale nell'aterosclerosi del tratto iliaco-femorale.

INTRODUCTION: Magnetic Resonance Angiography (MRA) potentials in all vascular districts have been largely applied also to peripheral vessels, where however there is no agreement as to the best type of sequence. We investigated the sensitivity, specificity and diagnostic accuracy of 2D TOF travel-sat MRA in the study of iliac-femoral artery stenoses. The gold standard was digital subtraction angiography (DSA). MATERIAL AND METHODS: Twenty patients (14 men and 6 women, mean age: 65 years with suspected atheromasic disease were examined. Diagnostic MRA and DSA were carried out within 48 hours of each other. A super-conductive 1 T magnet (Siemens Impact) and a body coil were used. 2D TOF travel-sat sequences were carried out with the following parameters: FA 40 degrees, TR 31 ms, ST 10 mm, overlap 1 mm, MA 128 x 512. The images acquired on the axial plane were postprocessed with MIP on the z axis from -15 degrees to 15 degrees. Two different radiologists evaluated MRA and DSA images and graded the stenoses on a multiple choice card: 1) negative, 2) 1-40%, 3) 41-70%, 4) 71-99%, 5) occlusion. RESULTS: MRA diagnosed 90 positives and 110 negatives: 60 were true positives, 92 true negatives, 30 false positives and 18 false negatives. MRA overestimated 8 cases and underestimated 4 cases. DSA findings were negative in 122 cases and positive in 78 cases: 4 grade 2, 10 grade 3, 28 grade 4 and 36 grade 5. Relative to DSA, MRA sensitivity, specificity and diagnostic accuracy were 72%, 75% and 74%, respectively; MRA diagnostic accuracy, sensitivity and specificity in hemodynamically severe stenoses (> 71%) were 80%, 78% and 71.8%, respectively. CONCLUSION: Our study confirms the usefulness of 2D TOF travel-sat MRA in the study of iliac-femoral stenoses.

[Magnetic resonance angiography in the study of pulmonary veins: TOF 3D versus 2D]. / Angiografia con Risonanza Magnetica nello studio delle vene polmonari: TOF 3D versus 2D.

The aim of this work was to optimize the magnetic resonance angiography (MRA) technique for the selective study of the pulmonary veins. Twenty consecutive patients (13 men and 7 women; mean age: 30.5 years) were examined. MRA was performed with a 1 T superconductive magnet and the 3D time of flight (TOF) technique. Fast sequences (3D FISP: TR 58 ms, TE 6 ms, FA 20 degrees, matrix 192 x 256; and 2D FLASH: TR 44 ms, TE 10 ms, FA 30 degrees, matrix 192 x 256) were used. Coronal and sagittal images were submitted to MIP processing; presaturation pulses for the pulmonary arteries were located in the mediastinal region. In the right lung, 3D TOF on the coronal plane well showed 124 veins, while sagittal images showed 106 veins. In the left lung, 3D TOF on the coronal plane well showed 96 vessels, while sagittal images showed 44 vessels. In the right lung, 2D TOF on the coronal plane well showed 54 veins, while sagittal images showed 36 vessels. In the left lung, 2D TOF on the coronal plane well showed 22 vessels, while sagittal images showed 21 vessels. Therefore, 3D TOF yielded better results than 2D TOF (p < 0.05). To conclude, 3D TOF with contrast agent administration is a useful tool to study the pulmonary veins; those with a larger caliber are better depicted and the integration of coronal and sagittal images depicts more veins.

Organic coronary stenosis in Prinzmetal's variant angina.

Functional factors are known to precipitate ischemic episodes at rest in variant angina, but the role of fixed coronary lesions is still debated. The prevalence, extent, severity and prognostic implications of organic coronary stenoses in variant angina were evaluated in 162 patients with transient ST segment elevation documented during hospitalization. Seventy-eight patients had normal coronary arteries or single-vessel coronary lesions (group 1) and 84 patients had multivessel coronary stenoses (group 2). Both groups were followed up for 82 +/- 41 months. Angiographically normal coronary arteries were observed in only 11 patients (7%). In 59 patients with single-vessel coronary stenoses, the internal luminal diameter was reduced by 51 +/- 12%. There were 20 deaths (16 from cardiac causes) during the 5-year follow-up. Kaplan-Meier survival analysis revealed a significantly lower 5-year survival rate in group 2 (80.1%) compared to group 1 (94.6%, p = 0.006 by Mantel-Haenszel test). If only cardiac causes of death were considered, the 5-year survival rate was still lower in group 2 (84.0%) than in group 1 (97.1%, p = 0.004). Considering both revascularized patients and those treated medically for the entire duration of the follow-up, the survival rate was significantly lower in group 2 than in group 1. Finally, the extent of coronary lesions was an independent predictor of survival by Cox multivariate regression analysis. Organic coronary stenoses are frequent in patients with variant angina and are important for the long-term prognosis.

Magnetic resonance angiography of the origins of the supraaortic arteries: comparison of single and double volume acquisition 3D time of flight.

PURPOSE: The authors have optimized the technique of Magnetic Resonance Angiography (MR-angio) in the study of the origins of the supraaortic arteries. METHODS: Twenty healthy volunteers, mean age 21.5, were studied with MR-angio of the origins of the supraaortic arteries; a 1 T superconductive magnet (Impact) with body coil was used. All the volunteers were studied using Time of flight (TOF), and two acquisition techniques: single volume FISP (fast imaging with steady-state precession) 3D TONE (titled optimized non-saturating excitation) and double volume FISP 3D TONE with 30% overlapping. The images were acquired on the axial plane following the positioning of three pre-saturation pulses. RESULTS: Regarding the visualization of the different components of the origins of the supraaortic arteries, a statistical analysis was worked out using the Mann Whitney test (p < 0.05); there was no statistically significant difference between two techniques. Regarding the visualization of the various segments (origin, 1/3 proximal, 1/3 middle, 1/3 distal), although the double volume did on the one hand allow a better visualization of the more distal vascular components (statiscally significant difference), on the other it induced the presence of artefacts with decreased the quality of the image as a whole. DISCUSSION: In our experience TOF 3D technique with TONE and single volume has well identified the origin of the vertebral arteries, while, in several cases, TOF 3D technique with TONE and double volume did not permit the visualization of the origin of the vertebral arteries due to the presence of the artefacts from overlapping. CONCLUSION: From our experience we have established that the TOF 3D technique with TONE and single volume makes it possible to obtain MR-angio of a high diagnostic quality in a short time (10-15 mn).

[Malignant pulmonary neoplasms: diagnostic possibilities of magnetic resonance angiography]. / Neoplasie maligne polmonari: possibilità diagnostiche dell'angiografia con Risonanza Magnetica.

The authors investigated the potentials of Magnetic Resonance Angiography (MRA) in the preoperative staging of malignant central lung neoplasms. Fifteen patients with malignant central lung neoplasms were submitted to MRA 48 hours before surgery, which was the gold standard. The patients were 7 women and 8 men; their mean age was 54.3 years (range: 39-65 years). MRA was performed with the 2D and 3D time-of-flight (TOF) technique. FLASH 2D sequences (FA 18 degrees, TR 40 ms, TE 10 ms, SL 4 mm) and FISP 3D sequences (FA 20 degrees, TR 30 ms, TE 3 ms, SL 1 mm) were used. The images were processed with the maximum intensity projection (MIP) and rotated on different planes with a 15 degrees step. A multiple choice card, implying preoperative reading by an MRA expert, was used; in the study of the relationship between neoplasms and contiguous vessels, only two diagnoses were possible--i.e., infiltrated, not infiltrated. This card included, in the preliminary phase, a code for each MRA pattern--i.e., the presence of a clear cleavage plane, vascular occlusion, stenosis, irregular vessel profile, turbulence and several of these patterns together. MRA was in agreement with surgery in 14/16 cases and overestimated two cases as infiltration. In our preliminary study, MRA had 100% sensitivity, 75% specificity and 87.5% diagnostic accuracy. To conclude, MRA proved to be a complementary tool to spin-echo MR sequences in the staging of malignant central lung neoplasms.

[Pulmonary artery: angiographic technique optimization with magnetic resonance]. / Arteria polmonare: ottimizzazione della tecnica di angiografia con risonanza magnetica.

The authors optimized the Magnetic Resonance Angiography (MRA) technique for the study of the different portions of the pulmonary artery-i.e., the common trunk, right and left main arteries and the lobar, segmental and subsegmental branches. Twenty volunteers were examined with MRA of the pulmonary arteries with a 1-T superconductive magnet (Impact) and the time-of-flight (TOF) technique. The volunteers were divided into two groups by age (ranges: 20-40 and 41-70 years). The trunk and main branches were studied with the 2D TOF technique, while 2D TOF images were compared with 3D TOF images in the study of peripheral (lobar, segmental and subsegmental) branches. The images were processed with the maximum intensity projection (MIP). In the statistical comparison between 2D and 3D TOF images, the t-test was used (p < 0.05). The 2D TOF images acquired on the sagittal oblique and coronal planes, with presaturation pulses, permitted optimal depiction of the trunk and of the right and left branches in all the volunteers. Both 2D and 3D TOF images yielded excellent images in the study of the lobar branches. In the volunteers under 40 years of age, 3D TOF sequences visualized the peripheral (segmental and subsegmental) vessels better than 2D TOF sequences (451 vs. 361); the difference was statistically significant. In the volunteers over 41 years of age, the ratio was reversed: 467 for 2D TOF vs. 362 for 3D TOF sequences; again, the difference was statistically significant. The correct integration of 2D and 3D TOF sequences, according to the different segments of the pulmonary artery and to patients' age, permitted the complete and accurate visualization of the pulmonary arteries with MRA.

[Magnetic resonance angiography of the origin of the supraaortic trunks: the tridimensional time-of-flight with single volume technique]. / Angiografia con risonanza magnetica dell' origine dei tronchi sovraaortici: tecnica tempo di volo tridimensionale con volume singolo.

This work was aimed at studying the origin of the epiaortic vessels with Magnetic Resonance Angiography (MRA). Twenty normal volunteers (mean age: 21.5 years) were examined with MRA with a superconductive magnet at 1 T (Impact). All volunteers were examined with a body coil and the time of flight (TOF) technique. The FISP 3D TONE sequence was used, with the following parameters: TR 46 ms, TE 10 ms, FA 20 degrees, ST 2.2 mm, matrix 192 x 256. The images, acquired on the axial plane with three presaturation pulses, were rotated according to the maximum intensity projection on the Z axis. To classify the images, a multiple choice card was used by a reader, with three diagnostic judgements for the different segments of the vessels (i.e., good, poor and not visualized). 3D TOF MRA permitted good visualization of the right and left common carotid in 80% and 77% of cases, of the right and left vertebral arteries in 55% and 50% of cases, of the left and right subclavian arteries in 47% and 62% of cases, and of the anonymous artery in 100% of cases. To conclude, our preliminary results indicate FISP 3D TOF MRA as the best noninvasive technique to study the origin of the epiaortic vessels.

[The peripheral circulation: ECG-gated magnetic resonance angiography]. / Circolo periferico: angiografia con Risonanza Magnetica con sincronizzazione con ECG.

This work was aimed at comparing magnetic resonance angiography (MRA) with and without ECG gating in the study of peripheral vessels. Ten volunteers, mean age 27.8 years, were examined with MRA of the femoral, popliteal and tibial segments. MRA was performed with a 1.5-T superconductive magnet, a transmit head coil and the TOF 2D technique. In all cases MRA was performed without cardiac gating and with different times of trigger delay (0, 20, 40, 70 and 200 ms). When comparing the different acquisitions, the number of vessels and the signal-to-noise (S/N) ratio were calculated. In the femoral segment, MRA without cardiac gating showed a mean of 7.7 vessels with 2.79 S/N ratio; MRA with 0 ms of trigger delay showed a mean of 13.1 vessels with 1.51 S/N ratio; MRA with 20 ms trigger delay showed a mean of 13.1 vessels with 1.52 S/N ratio; MRA with 40 ms trigger delay showed a mean of 13.2 vessels with 1.52 S/N ratio; MRA with 70 ms trigger delay showed a mean of 13.5 vessels with 1.50 S/N ratio; finally, MRA with 200 ms trigger delay showed a mean of 13.4 vessels with 1.50 S/N ratio. In the popliteal segment, the corresponding values were 6.4 vessels and 2.51 S/N ratio, 11.4 vessels with 1.54 S/N ratio, 11.3 vessels and 1.54 S/N ratio, 11.6 vessels and 1.52 S/N ratio, 11.8 vessels and 1.52 S/N ratio and, finally, 11.9 vessels and 1.52 S/N ratio. In the tibial segment, the corresponding values were 8.5 vessels and 1.84 S/N ratio, 14.4 vessels and 1.14 S/N ratio, 14.5 vessels and 1.17 S/N ratio, 14.5 vessels and 1.14 S/N ratio, 14.3 vessels and 1.17 S/N ratio and, finally, 14.5 vessels and 1.19 S/N ratio. To conclude, MRA with cardiac gating better visualized peripheral vessels whatever the trigger delay.

Complex coronary artery lesion morphology influences results of stress echocardiography.

BACKGROUND: The likelihood of a positive response with dipyridamole stress echocardiography (DSE) is directly related to the extent and severity of angiographically assessed coronary artery disease. Whether coronary lesion morphology--a known predictor of adverse cardiac events--may also modulate stress echo results remains unknown. The objective of our study was to assess the relation between stenosis lesion morphology and stress echocardiographic results. METHODS AND RESULTS: High-dose (up to 0.84 mg/kg over 10 minutes) DSE and coronary angiographic data of 68 in-hospital patients (39 with stable angina, 29 with angina at rest) with nonoccluding, single-vessel disease at angiography and no previous myocardial infarction were analyzed. DSE was performed in all patients within 3 days of coronary angiography. An angiographic lesion was considered complex when irregular borders and/or intraluminal lucencies suggestive of ulcer and/or thrombus were present. According to angiographic lesion morphology, two groups were identified: group 1, with simple coronary lesions, and group 2, with complex coronary lesions. The two groups were matched for number of patients (n = 34 in each group), age (group 1, 59 +/- 9 versus group 2, 59 +/- 10 years, P = NS), and coronary artery stenosis severity by quantitative coronary angiography (group 1, 60 +/- 7% versus group 2, 58 +/- 6% diameter reduction, P = NS). The sensitivity of DSE was lower in patients of group 1 when compared with group 2 (53% versus 85%, P < .001). Among positive DSE, the low-dose (0.56 mg/kg over 4 minutes) positivity was less frequent in group 1 than in group 2 patients (17% versus 62%, P < .01). Exercise ECG was completed in 66 patients, and it was positive (> .1 mV ST-segment shift from baseline) in 20 out of 33 group 1 and in 22 out of 33 group 2 patients (61% versus 67%, P = NS). The peak rate-pressure product tended to be higher in group 1 than in group 2 patients (257 +/- 52 versus 240 +/- 64 mm Hg x beats per minute x 10(2), P = NS). CONCLUSIONS: In patients with single-vessel disease without coronary occlusion or previous myocardial infarction, coronary lesion morphology of the complex type is associated with a higher DSE sensitivity and with a greater prevalence of low-dose, positive responses. Presence of irregular plaque contours, not only plaque geometry, is important in modulating stress responses in the presence of angiographically assessed coronary artery disease.

ANP kinetics in normal men: in vivo measurement by a tracer method and correlation with sodium intake.

125I-labeled atrial natriuretic peptide (ANP) was bolus injected into seven healthy human male subjects on an unrestricted diet (sodium intake ranging from 80 to 300 mmol/day). A high-performance liquid chromatographic procedure was used to purify the labeled hormone and the principal labeled metabolites in venous plasma samples collected up to 50 min after injection. The main ANP kinetic parameters were derived from the disappearance curves of the 125I-ANP, which were satisfactorily fitted by a biexponential function in all subjects. Newly produced ANP initially distributes in a large space (plasma-equivalent volume is 12.1 +/- 3.6 l/m2 body surface); the hormone rapidly leaves this sampling space through both degradation and distribution in peripheral spaces, as indicated by the single-pass mean transit time through the sampling space (3.9 +/- 1.2 min). The mean residence time in the body (22.7 +/- 23.1 min) and the plasma-equivalent total distribution volume (30.9 +/- 12.0 l/m2) indicate that ANP is also widely distributed outside the initial space. Metabolic clearance rate (MCR) values were distributed across a wide range (from 740 to 2,581 ml.min-1 x m-2) and were shown to correlate strongly with the daily urinary excretion of sodium. These results indicate that: 1) newly produced ANP is rapidly distributed and degraded, 2) the body pool of the hormone can be considered as a combination of two exchanging spaces, 3) circulating ANP is < or = 1/15 of the body pool, and 4) MCR of ANP is closely related to sodium intake, at least in normal subjects on a free sodium intake diet.

Histologic evidence of myocardial damage in apparently healthy subjects with ventricular arrhythmias and myocardial dysfunction.

The association of ventricular arrhythmias and myocardial dysfunction could be considered an early step toward cardiomyopathy; therefore, we studied 28 patients in NYHA class I and II, characterized by complex ventricular arrhythmias (VA) on 24-h Holter monitoring and volumetric and/or contractile abnormalities on a standard two-dimensional echocardiogram (2-D echo). All patients underwent radioisotopic angiography, 20 patients complete hemodynamic study, and 15 patients endomyocardial biopsy. Ambulatory ECG monitoring showed the presence of frequent premature ventricular contractions in 14 patients (50%) and episodes of ventricular tachycardia in 16 patients (57%). 2-D echo showed mono- or biventricular enlargement and dyssynergies in 25 patients (89%) (left ventricle in 6, right ventricle in 11, both in 8). Two patients showed only left ventricle enlargement and one patient isolated left ventricular dyssynergies. Radioisotopic angiography showed mono- or biventricular ejection fraction reduction in 24 patients (85%) and regional dyssynergies in 24 patients (85%) in accordance with 2-D echo. Hemodynamic study showed in all patients normal coronary arteries, and right and left angiography confirmed enlargement and/or regional dyssynergies. Endomyocardial biopsy was abnormal in 11 of 15 patients: various degrees of hypertrophy, parcellar fibrosis, and adipogenic infiltration were found. Our preliminary data suggest that the simultaneous occurrence of ventricular arrhythmias and ventricular dyssynergies and/or enlargement in patients without apparent clinical heart disease may represent an early stage of dilated cardiomyopathy.

Atrial natriuretic peptide during different pacing modes in a comparison with hemodynamic changes.

The study investigates the response of atrial natriuretic peptide (ANP) to different cardiac pacing modes in comparison with hemodynamic changes. Ten patients underwent Swan-Ganz catheterization during pacemaker implant. Atrioventricular and ventricular pacing were performed consecutively at three pacing rate levels (80, 100, and 110 ppm). Blood samples were taken from the pulmonary artery for ANP determination, both basally and at the end of each pacing period. Concomitantly, mean pulmonary capillary wedge pressure (PCWP) and mean pulmonary artery pressure (PAP) were measured. Cardiac output (CO) was determined by thermodilution both basally and during the 110 ppm steps. During atrioventricular pacing, whereas no significant changes were observed for ANP, PCWP and PAP, CO increased significantly (P less than 0.0005). At the beginning of ventricular pacing hemodynamic parameters and ANP levels were comparable with those of baseline conditions. During subsequent ventricular pacing PCWP and ANP increased significantly at the 110 ppm rate step (P less than 0.05). PAP did not change significantly, whereas CO decreased in all cases (P less than 0.01). A positive correlation was observed between ANP and PCWP during ventricular (P less than 0.001), but not atrioventricular pacing. The results, while confirming the hemodynamic advantages of atrioventricular pacing, point to a major stimulation of ANP secretion during ventricular pacing. This fact, together with the observed drop in CO and the correlation between ANP and PCWP, suggest that the increase of ANP in ventricular pacing may be the expression of a compensatory mechanism to the hemodynamic disadvantages of atrioventricular asynchrony.