In clinical practice and in clinical trials, echocardiography and scintigraphy are used the most for the evaluation of global left ejection fraction (LVEF) and left ventricular (LV) volumes. Actually, poor quality imaging and geometrical assumptions are the main limitations of LVEF measured by echocardiography. Contrast agents and 3D echocardiography are new methods that may alleviate these potential limitations.Therefore we sought to examine the accuracy of contrast 3D echocardiography for the evaluation of LV volumes and LVEF relative to MIBI gated SPECT as an independent reference. In 43 patients addressed for chest pain, contrast 3D echocardiography (RT3DE) and MIBI gated SPECT were prospectively performed on the same day. The accuracy and the variability of LV volumes and LVEF measurements were evaluated.Due to good endocardial delineation, LV volumes and LVEF measurements by contrast RT3DE were feasible in 99% of the patients. The mean LV end-diastolic volume (LVEDV) of the group by scintigraphy was 143 ± 65 mL and was underestimated by triplane contrast RT3DE  and less by full-volume contrast RT3DE . Limits of agreement with scintigraphy were similar for triplane andfull-volume, modalities with the best results for full-volume. Results were similar for calculation of LV end-systolic volume (LVESV). The mean LVEF was 44 ± 16% with scintigraphy and was not significantly different with both triplane contrast RT3DE (45 ± 15%) and full-volume contrast RT3DE (45 ± 15%). There was an excellent correlation between two different observers for LVEDV, LVESV and LVEF measurements and inter observer agreement was also good for both contrast RT3DE techniques.Contrast RT3DE allows an accurate assessment of LVEF compared to the LVEF measured by SPECT, and shows low variability between observers. Although RT3DE triplane provides accurate evaluation of left ventricular function, RT3DE full-volume is superior to triplane modality in patients with suspected coronary artery disease. The determination of the global LV function and cardiac volumes remains one of the most important issues in the everyday practice of the cardiologist because these measurements are shown to correlate most strongly with prognosis among patients with cardiac disease ,2. Due tRT3DE technology, which has recently become widely available, allows fast acquisition from a single acoustic window of dynamic data that encompass the entire heart. Using 3D imaging, less geometrical assumptions are made, and thus RT3DE should be more useful for the assessment of left ventricular size and function. Several recent studies have demonstrated the potential improvements in the evaluation of global LV function from RT3DE data . One impTherefore, we aimed to compare the feasibility and the accuracy of contrast enhanced RT3DE triplane and RT3DE full-volume with MIBI gated SPECT, as an independent reference, for the evaluation of the LVEF and cardiac volumes.We prospectively recruited 43 consecutive patients addressed for chest pain to the echocardiography laboratory for measurement of LV volumes and LVEF. After exclusion of one patient with poor echocardiographic images and three with atrial fibrillation, a study group of 38 patients  who underwent contrast RT3DE and MIBI gated SPECT during the same day within one hour.Enhanced contrast studies were performed with a commercially available echocardiographic platform , equipped with a 3V-probe for triplane acquisition in harmonic mode. Patients were scanned in the left lateral decubitus position, from the apical window. Care was taken to visualise the true LV apex allowing simultaneous acquisition of the apical four-, two- and three-chamber views. Figure  CineloopRT3DE full-volume images with contrast were also obtained from an apical window with the patient in the same position. Images were gathered over 4 cardiac cycles using the same matrix-array ultrasonographic transducer. Measurements of RT3DE volumes were performed offline as previously described   and comp®  is an aqueous suspension of stabilized SF6 microbubbles. The size of these microbubbles is between 1 and 10 μm, and their number is between 2 and 5 × 108 per mL. The solution was prepared according to the manufacturer's instructions and injected using a dedicated pump  at an initial rate of 0.5 ml/min. The rate of bolus infusion was adapted for optimal visualization of endocardial border definition. The contrast-enhanced imaging was performed using predefined settings .The commercial available contrast agent SonovueLVEF was calculated from the gated SPECT images using the full automatic program developed at Cedars-Sinai .Myocardial perfusion SPECT gated in eight time bins were acquired 60 min after the injection of 925 MBq technetium-99m labelled sestamibi (MIBI) at rest using a dual-head gamma camera  Detailed imaging and data processing have been reported previously . The sofData are presented as mean ± SD. Two-tailed t-tests were used where appropriate. Linear regression with Pearson's correlation coefficient was used to assess correlations between RT3DE and MIBI gated SPECT data. Bland-Altman analysis was used to express agreement between RT3DE and gated-SPECT measurements. A subgroup of 20 randomly selected patients was studied for interobserver variability, which was determinated by using the same set of contrast RT3DE images measured by two separate sonographers. The interobserver variability in quantitative RT3DE LV volumes and LVEF was assessed by calculating the linear regression with Pearson's correlation coefficient and the limits of agreement between measurements made by use of Bland-Altman analysis between the two different observers . A valueThe baseline characteristics of the patients are summarized in Table The mean LVEDV of the group by scintigraphy was 143+65 mL (range: 66–323 mL) and was underestimated by triplane contrast RT3DE  and less by full-volume contrast RT3DE . Limits of agreement with scintigraphy were similar for triplane andfull-volume modalities with the best results for full-volume (mean of the difference ± 2 SD: 5.2 ± 12.2). Results were similar for calculation of LVESV. By scintigraphy, the mean LVESV was 88 ± 62 mL (range: 26–264 mL) versus 75 ± 54 mL with triplane (p < 0.001) and 80 ± 57 mL with full-volume (p < 0.001). The mean LVEF was 44 ± 16% with scintigraphy and was not significantly different with both triplane contrast RT3DE (45 ± 15%) and full-volume contrast RT3DE (45 ± 15%).The correlation between contrast RT3DE triplane and full-volume with gated SPECT and the Bland-Altman plots are shown in Figure 2 = 0.9; p < 0.0001), LVESV  and LVEF  respectively. Inter-observer agreement was also good for LVEDV (mean of the difference ± 2 SD: -0.09 ± 6.1), LVESV (mean of the difference ± 2 SD:-0.3 ± 7.8) and LVEF (mean of the difference ± 2 SD:-0.8 ± 7).Triplane contrast RT3DE has shown a good correlation between two different observer's measurements for LVEDV , LVESV  and LVEF  respectively. Inter observer agreement was also good LVEDV, LVESV and LVEF .Regarding full-volume contrast RT3DE, there was also an excellent correlation between two different observer's measurements for LVEDV . Given that the images were obtained at rest, the temporal resolution of contrast RT3DE was similar to that obtained with MRI and superior compared to gated SPECT.On another hand, full-volume dataset may be insufficient to capture the entire LV volume of a large heart but this might be alleviated by ongoing technical improvements."Compared to a previous study  for a grThe evaluation of LV volumes and LVEF is sought for major decisions in cardiology. The combination of contrast with RT3DE offers the unique opportunity to alleviate the main limitations of 2DE and to be used in unselected population. RT3DE does not have significant limitations in clinical application such as exposure to ionizing radiation (the dose of a technetium sestamibi scan corresponds to 500 chest x rays and to an extra risk of cancer of about 1 in 2000 exposed patients for one single examination), exposure to radiographic contrast material, cost, invasive nature, or contraindications such as the presence of implanted metallic objects or claustrophobia. Moreover, contrast RT3DE has the advantage to be a portable and versatile technique with a high spatial and temporal resolution that can be performed at bedside. Therefore it is the ideal tool making serial study of LV volumes and LVEF practical.Contrast was used in all patients without any selection. Even patients with good quality image had received contrast. We did not analyze the impact of contrast in function of baseline image quality. Therefore, the requirement for contrast for all RT3DE studies can not be extrapolate from the present study. However, it has been shown that increase of inter-observer reproducibility with contrast enhanced left ventricular opacification was not restricted to patients with poor baselineimage quality (11), so the benefit of contrast may not completely relate to improved endocardial resolution. The avoidance of off-axis image projections with contrast enhanced left ventricular opacification in patients with good resting images likely explains the better accuracy of LV and LVEF calculations with RT3DE.The study patients were all in sinus rhythm; thus, the results cannot be generalized to patients with arrhythmia. The reason for excluding these patients was mainly the risk of inadequate gated SPECT quality.Contrast 3D echocardiography allows an accurate assessment of LVEF compared to the LVEF measured by SPECT, and is reproducible. Although RT3DE triplane provides accurate assessment of left ventricular function, RT3DE full-volume is superior to triplane modality. However, both methods have sufficient accuracy to provide a close approximation for a valuable measurement of volumes and LV function compared to gated SPECT in patients with suspected coronary artery disease. This technique provides a quick, inexpensive, portable and accurate alternative for LV size and function analysis and can be considered as the method of choice for the daily measurement of LVEF, especially when serial measurements are required.The authors declare that they have no competing interests.BC, DH and SD participated in the design of the study and performed the statistical analysis. BC and PL conceived of the study, and participated in its design and coordination. SW, PM, BC and EL participated to the acquisition and the analysis of the images. MM and SH participated to the elaboration of the manuscript. All authors read and approved the final manuscript.Contrast enhanced triplane real time 3D echocardiography. Example of contrast triplane real time 3D echocardiography.Click here for fileContrast enhanced full volume real time 3D echocardiography. Example of 9 slices contrast full volume real time 3D echocardiography.Click here for file


The study aimed to determine the frequency and characteristics of heart failure with normal EF in a native African population with heart failure.It was a hospital cohort study. Subjects were 177 consecutive individuals with heart failure and ninety apparently normal control subjects. All the subjects underwent transthoracic echocardiography. The group with heart failure was further subdivided into heart failure with normal EF (EF ≥ 50) (HFNEF) and heart failure with low EF(EF <50)(HFLEF).The subjects with heart failure have a mean age of 52.3 ± 16.64 years vs 52.1 ± 11.84 years in the control subjects; p = 0.914. Other baseline characteristics except blood pressure parameters and height were comparable between the group with heart failure and the control subjects. The frequency of HFNEF was 39.5%. Compared with the HFLEF group, the HFNEF group have a smaller left ventricular diameter (in diastole and systole):  respectively, a higher relative wall thickness and deceleration time of the early mitral inflow velocity: (0.4 ± 0.12 vs 0.3 ± 0.14 p < 0.0001 and 149.6 ± 72.35 vs 110.9 ± 63.40 p = 0.001) respectively.The two groups with heart failure differed significantly from the control subjects in virtually all echocardiographic measurements except aortic root diameter, LV posterior wall thickness(HFLEF), and late mitral inflow velocity(HFNEF). HFNEF accounted for 70(39.5%) of cases of heart failure in this study.Hypertension is the underlying cardiovascular disease in 134(75.7%) of the combined heart failure population, 58 (82.9%) of the subjects with HFNEF group and 76(71%) of the HFLEF group. Females accounted for 44 (62.9%) of the subjects with HFNEF against 42(39.3%) in the HFLEF group (p = 0.002).The frequency of heart failure with normal EF in this native African cohort with heart failure is comparable with the frequency in other populations. These groups of patients are more likely female, hypertensive with concentric pattern of left ventricular hypertrophy. Congestive heart failure has been recognized from ancient times . The facIt is recognised that there is a group of patients presenting with the syndrome of congestive cardiac failure in the setting of a normal ejection fraction,13. The Although this entity has been challenged on the basis of the presentation and symptomatology which is at least similar to the features of other conditions common in the elderly and, cardiovascular changes associated with aging ,16, studFrom previous studies, it is known that patients with heart failure and normal ejection fraction are typically elderly, often females, usually obese and frequently have hypertension and diabetes . This stThe study was carried out at the cardiology unit of the department of medicine of the University College Hospital, Ibadan, Nigeria. The University College Hospital Ibadan is the only tertiary health care facility located in the city of Ibadan in south western Nigeria with more than three million inhabitants.Eligible patients were adult men and women aged 18 years and above, presenting with syndrome of heart failure. A total of 205 consecutive subjects with heart failure were screened. 177 subjects were found eligible after twenty one subjects were excluded on account of suboptimal echocardiographic image qualities and seven subjects on account of co-existent chronic obstructive airway disease based on history or clinical examination or chest radiograph. A second group consisted of ninety apparently normal men and women who had no clinical history of cardiovascular or any other medical condition. They were recruited from the medical  and non-medical staff of the University College Hospital Ibadan and patients' relations. The two groups were comparable in their age and sex ratios.Informed consent was obtained from all the study participants before enrolment into the study. Ethical clearance was obtained from the joint University of Ibadan and University College Hospital ethical committee.Diagnosis of congestive heart failure was based on the Framingham's criteria for diagnosis of heart failure. Major cSubjects were weighted without shoes and in light clothing on a standard beam balance. Height was measured to the nearest centimetre using anthropometrical plane with subjects not putting on shoes or headgear.2 Body surface area was calculated using the formula of Dubois[Body mass index (BMI) was calculated using the formula BMI = Weight (kg)/(height)of Dubois.Diagnosis of heart failure with normal ejection fraction required:(1) Minimum of two major criteria or one major criterion plus two minor criteria.(2) Left ventricular ejection fraction (LVEF) equal to or more than 50%.Heart failure with reduced/low ejection fraction required (1) above and LVEF < 50%.Hypertension was deemed present if systolic BP ≥ 140 mmHg and or diastolic BP ≥ 90 mmHg,23 on atThe BP was recorded in the right arm of subjects who have rested for at least 5 minutes with an appropriately sized cuff.Coronary artery disease was considered present and a cause of heart failure when there was evidence of definite myocardial infarction from history, ECG or echocardiogram or if there is a long history of stable angina pectoris.Valvular heart disease was considered the primary cause of heart failure when there was echocardiographic evidence of valvular heart disease of more than moderate severity.Dilated cardiomyopathy was diagnosed based on dilated cardiac chambers with increased left ventricular mass and normal wall thicknessEchocardiogram was also employed to exclude pericardial and congenital heart diseases.Echocardiography assessment was carried out with ALOKA SSD - 1700 echo machine which was equipped with a 3.5 MHz linear array transducer. Two dimensional guided M-mode echocardiography was performed on each subject in the left lateral decubitus position. All measurements were made according to the American Society of Echocardiography leading edge to leading edge criteriaAverage oLV internal diameter and interventricular septal and posterior wall thickness was measured at end diastole and end systole. Left ventricular (LV) systolic performance was assessed using the fractional shortening of the left ventricle and the ejection fraction.LV ejection fraction was calculated using the Teichholz calculation formula.Fractional shortening was calculated from LV internal dimensions in diastole and systoleLeft ventricular mass(LVM) was calculated using the Devereux-modified ASE cube formular.Where, VSTd is the ventricular septal thickness in diastole; PWTd is the posterior ventricular wall thickness in diastole and LVIDd is the left ventricular internal diameter in diastoleRelative wall thickness(RWT) was calculated using.The aortic root was measured at end diastole and the left atrium was measured at end-ventricular systole, including the thickness of the posterior wall of the aortaLeft ventricular diastolic function was evaluated by studying the filling dynamics of the left ventricle. The mitral inflow velocities were measured from the apical four chamber view with pulsed-wave Doppler and with the sample volume positioned at the tip of the mitral valve leaflets. This inflow characteristics were quantified by measuring the transmitral "E" wave velocity  and the "A" wave velocity , the E/A ratio and the deceleration time (DT): -31. Two Statistical analysis was performed using SPSS software version 10.0. Means were expressed as means (standard deviation) while proportions were expressed as count (percentages). Comparison of categorical variables between the groups was by the chi square test, while continuous variables were compared using the students't' test for independent groups. Analysis of variance was used to comparison of mean between 3 or more groups. Non parametric tests were used where appropriate. A two tailed p value < 0.05 was considered to be significant.Table Stratifying the subjects with heart failure into 2 groups based on whether the ejection fraction is preserved or reduced and comparing with control subjects . Otherwise measures of transmitral flow are comparable between the 2 groups. Compared with the control subjects, both groups have higher early mitral inflow velocity (E) and higher ratio of early inflow velocity to the atrial mitral inflow velocity (E/A ratio) and a lower deceleration time of early transmitral flow velocity (E). Additionally, the group with reduced EF have a lower late transmitral flow velocity (A).The study showed that patients presenting in heart failure are more likely to have a higher left ventricular mass, thicker septum and more dilatation of the left atrium and the left ventricle when compared with normal subjects. It also showed that subjects with heart failure and preserved ejection fraction and subjects with reduced EF have comparable left ventricular wall thickness and left atrial dilatation. The major differences between these 2 groups are more LV dilatation and reduced deceleration time of E in the subjects with reduced EF. Heart failure with normal ejection fraction occurred in one out of every three heart failure patients. Hypertension was the most frequent underlying cardiovascular disease associated with heart failure and also heart failure with preserved ejection fraction in this African population of subjects with heart failure.In the study the mean age of patients with heart failure was 52.3 ± 16.6 years which is comparable with the findings in previous studies by Falase et al over 2 dThe mean age of the group presenting with heart failure and normal ejection fraction and the group presenting with reduced ejection fraction was not significantly different. This finding is similar to previous studies in Caucasian populations by Varela-Roman et al and SennThe systolic blood pressure and pulse pressure were significantly different between the group with normal ejection fraction and the group with reduced ejection fraction. These parameters were higher in the former. These findings are consistent with the fact that hypertension was found to be a more frequent aetiology of heart failure in the group with normal ejection fraction than in the group with reduced ejection fraction. Also, a narrow pulse pressure is an expected accompaniment of the low ejection fraction characterising heart failure with low EF. Ahmed et al  had demoComparing the heart failure groups, it was found that the end diastolic diameter, end systolic diameters were significantly larger in the group with low ejection fraction, while expectedly the relative wall thickness was higher in the group with preserved ejection fraction. Distinct relationship between left ventricular dilatation and poor systolic function is well established. Devereux et al and TsutThe shorter deceleration time(DT) in the group with heart failure and reduced ejection fraction is similar to the findings by Devereux et al. This isHypertension was the overall leading cause of heart failure in this study. Furthermore hypertension was implicated even more frequently in patients with preserved ejection fraction. This is consistent with the findings in some other populations ,41. The Although there is considerable debate whether HFNEF can evolve into HFLEF, we are of the view that in Africa where hypertension is the main aetiology, this may be the case. Some studies have suggested this in other populations and there is need to study the natural history HFNEF in native Africans -45.The study, a hospital based, carried out in a single centre might not be completely representative of the findings in the community or in other centres, hence a multicentred and/or community based study would be desirable to fully characterise heart failure with preserved EF in this population group.Use of other modalities of assessing diastolic function such as pulmonary venous flow velocities, tissue Doppler echocardiography or invasive measurements could have helped to further establish and characterise diastolic dysfunction in this study population. These were not included in the study protocol.We also recognise the use of Teicholtz formula rather than the modified Simpson's method in the assessment of the ejection fraction (in the presence of ventricular dilatation) as a limitation of the studyHeart failure with normal ejection fraction constitutes a significant part of the heart failure burden in this native African cohort and the frequency of 39.5% found in this study is comparable with findings in other populations. Hypertension was the most common underlying cardiovascular condition overall and in the different heart failure groups, the highest relative frequency being in the group with heart failure and normal ejection fraction. Patients with heart failure and normal ejection fraction are more likely female with higher body mass index, wider pulse pressure, thicker septum, and a more concentric pattern of hypertrophy when compared with the group with reduced ejection fraction.The authors declare that they have no competing interests.AKA, OOO, AAA, AOF participated in the original design of the study. OSO, AA, and DBO participated in the collection of data. AKA and OSO drafted the manuscript. All authors contributed in the interpretation of the study data and have seen and approved the final version of the report.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/9/52/prepub


Renalase is a soluble enzyme that metabolizes circulating catecholamines. A common missense polymorphism in the flavin-adenine dinucleotide-binding domain of human renalase (Glu37Asp) has recently been described. The association of this polymorphism with cardiac structure, function, and ischemia has not previously been reported.We genotyped the rs2296545 single-nucleotide polymorphism (Glu37Asp) in 590 Caucasian individuals and performed resting and stress echocardiography. Logistic regression was used to examine the associations of the Glu37Asp polymorphism  with cardiac hypertrophy (LV mass>100 g/m2), systolic dysfunction (LVEF<50%), diastolic dysfunction, poor treadmill exercise capacity (METS<5) and inducible ischemia.Compared with the 406 participants who had GG or CG genotypes, the 184 participants with the CC genotype had increased odds of left ventricular hypertrophy , systolic dysfunction , diastolic dysfunction , poor exercise capacity , and inducible ischemia . The Glu37Asp (CC genotype) caused a 24-fold decrease in affinity for NADH and a 2.3-fold reduction in maximal renalase enzymatic activity.A functional missense polymorphism in renalase (Glu37Asp) is associated with cardiac hypertrophy, ventricular dysfunction, poor exercise capacity, and inducible ischemia in persons with stable coronary artery disease. Further studies investigating the therapeutic implications of this polymorphism should be considered. The kidney, in addition to maintaining fluid and electrolyte homeostasis, performs essential endocrine functions Human renalase is encoded by a 311Kbp gene with 10 exons located on chromosome 10q23.33. The renalase protein is well conserved with orthologs present in chimpanzees (95% amino acid identity) and cyanobacteria (23% identity). The major isoform of renalase contains 342 amino acids comprising a signal peptide (amino acids 1–17), a flavin-adenine dinucleotide (FAD) binding domain (amino acids 4–45), and a monoamine oxidase domain (amino acids 75–342). Evidence exists for at least four alternatively-spliced isoforms of renalase Whether common genetic variation at this locus affects cardiac structure, function, and ischemia in humans is not known. We therefore genotyped the (Glu37Asp) polymorphism and evaluated its association with cardiovascular phenotypes in a large cohort of individuals with stable coronary artery disease.The Heart and Soul Study is a prospective cohort study investigating the influence of psychosocial factors on cardiovascular events in outpatients with stable coronary artery disease. The enrollment process for the Heart and Soul Study has been previously described The study protocol was approved by the following Institutional Review Boards: the University of California San Francisco Committee on Human Research, the Research and Development Committee at the San Francisco VA Medical Center, the Medical Human Subjects Committee at Stanford University, the Human Subjects Committee at the VA Palo Alto Health Care System, and the Data governance Board of the Community Health Network of San Francisco. All participants provided written informed consent. Between September 2000 and December 2002, a total of 1024 participants enrolled in the study. Of these, DNA samples were available for analysis in 982 individuals, including 590 of Caucasian ethnicity. There were no demographic differences between individuals who did and those who did not provide DNA samples for analysis.Prior to the study appointment, participants completed an overnight fast except for taking their regularly prescribed medications. Fasting venous blood samples were drawn into chilled ethylene-diamine-tetra-acetic acid tubes, and peripheral blood leukocytes were stored at −70° Celsius. Samples were later thawed, and genomic DNA was extracted using a salt modification method (Gentra Systems). The ABI PRISM 7900HT Sequence Detection System was used to perform genotyping of the rs2296545 single nucleotide polymorphism using the 5′nuclease allelic discrimination assay . The assay kit included the forward and reverse target-specific polymerase chain reaction primers, and the Taqman MGB probes labeled with dyes DFAM and VIC. The total volume of the sequencing reaction was 5-ul containing 2.5ul of Taqman Universal PCR Master Mix, 0.083ul of 40X Taqman MGB Assay Mix, 1.417ul of ddH2O, and 1ul (5 ng) of genomic DNA. Investigators blinded to the clinical data performed the genotyping assays.2.All participants underwent complete resting 2-dimensional echocardiography and Doppler examination using an Acuson Sequoia ultrasound system  with a 3.5-MHz transducer. Standard parasternal short-axis and apical 2 – and 4-chamber views were obtained and planimetered to determine end-diastolic and end-systolic volumes. The left ventricular ejection fraction (LVEF) was calculated as (end diastolic volume – end systolic volume)/end diastolic volume. Systolic dysfunction was defined as LVEF<50%. Diastolic dysfunction was defined as the presence of one of the following: pseudonormal transmitral inflow pattern defined as a ratio of peak mitral early diastolic to atrial contraction velocity (E/A) of 0.75<E/A<1.5 with diastolic dominant pulmonary vein flow; restrictive filling defined as an E/A of 1.5 or greater with diastolic dominant pulmonary vein flow All participants then underwent a symptom-limited graded exercise treadmill test according to a standard Bruce protocol. To achieve maximal heart rate, participants who were unable to continue the standard Bruce protocol were switched to lower manual settings on the treadmill and encouraged to exercise for as long as possible. Maximal exercise capacity in metabolic equivalents (METS) was determined at peak exercise. Poor exercise capacity was defined as<5 METS at peak exercise 2). All participants were instructed to bring their medication bottles to the study appointment where study personnel recorded all current medications. Medications were categorized using Epocrates Rx , an electronic reference database that contains 3300 drugs classified by both generic and commercial product names. Fasting serum chemistry samples were used to measure triglycerides, HDL cholesterol, LDL cholesterol. Serum creatinine was measured by the rate Jaffe method. The intra-individual coefficient of variation was 2%. Estimated glomerular filtration rate (GFR) was calculated by the abbreviated (4-variable) Modification of Diet and Renal Disease Study formula as follows: estimated GFR = 186x(serum creatinine−1.154)x(age−0.203)xx(1.21 if black) Baseline demographics, age, sex, and ethnicity were determined by self-reported questionnaire. Cardiovascular co-morbidities including hypertension, diabetes, hyperlipidemia, prior myocardial infarction, prior congestive heart failure, prior stroke, smoking status, and prior revascularization were determined by self-report. Recumbent blood pressure was measured after 5 minutes at rest. Participants were weighed and measured without shoes. Body Mass Index (BMI) was calculated as the ratio of mass (kg) to the square of height . E. coli BL21 were transformed and grown at 37°C for 16 hours with 0.1 µM flavin adenine dinucleotide (FAD). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added for the last 3.5 hours of culture. Recombinant renalase was purified from inclusion bodies and refolded as previously described −1 cm-. Background correction was achieved by subtracting the changes in absorbance obtained with BSA. To estimate kinetics parameters, initial velocity was plotted against NADH concentration, and the data were fitted to the Michaelis-Menten equation using non-linear regression .Since renalase is a NADH/FAD dependent oxidoreductase, the enzymatic activity the each variant was assessed by measuring the rate of reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5--2H tetrazolium monosodium salt (WST-1) as a function of NADH concentration. Reduction of WST-1 results in the formation of a yellow, water-soluble formazan product, which can be quantified by measuring absorbance at 450 nm Differences in baseline characteristics between participants with distinct rs2296545 genotypes  were compared with the use of analysis of variance for continuous variables and the chi-squared test for dichotomous variables. We then examined differences in cardiac phenotype by genotype using generalized linear models for continuous measures and logistic regression for dichotomous measures of cardiac function. Participants with GG and GC genotypes were combined for multivariable analyses because they had similar cardiac function in unadjusted analyses. Multivariable adjustment was made for age, gender, BMI, systolic blood pressure, diastolic blood pressure, and estimated GFR. Covariates were selected on their potential for confounding or mediating effects. Model assumptions were checked by visual inspection of directed acyclic graphs Statistical analysis was performed using SAS software version 9.1 . The authors take responsibility for the integrity of the data. All authors had full access to the data, except N.B.S. who was blinded to the genotype data. All authors have read and agree to the manuscript as written.We genotyped 590 Caucasian subjects in the Heart and Soul Study for the rs2296545 (Glu37Asp) single-nucleotide polymorphism. The genotype distribution was GG (107), CG (299), and CC (184). The observed frequencies of the G and C alleles were 0.43 and 0.57 respectively. These findings were consistent with Hardy-Weinberg equilibrium (p = 0.99). In a Han Chinese population, the observed frequencies of the G and C alleles were 0.39 and 0.61 in hypertensive individuals, and 0.44 and 0.56 in controls respectively .Parameters of cardiovascular structure, function, and ischemia categorized by genotype at rs2296545 are shown in . The proportion of participants with abnormal cardiovascular phenotype classified by genotype at rs2296545 is shown in . We found no evidence that the association of renalase genotype with cardiovascular structure and function was modified by age .In logistic regression models adjusted for age, demographics, BMI, systolic and diastolic blood pressure, and estimated GFR, the CC genotype was associated with left ventricular hypertrophy , systolic dysfunction , diastolic dysfunction , poor exercise capacity , and inducible ischemia  . The pro.Renalase is a FAD dependent oxidoreductase, which uses NADH as a cofactor to reduce its FAD moiety. In the presence of oxygen the reduced renalase-FADH complex metabolizes subtrates such as catecholamines. Renalase has intrinsic NADH oxidase activity since, in the absence of substrates, it can convert NADH to NAD+ and generate superoxide anion as a byproduct. To test if the Glu37Asp polymorphism had any detectable functional consequence, we compared the NADH oxidase activity of the Glu37 and Asp37 variants of the recombinant renalase by measuring the rate of reduction, of the electron acceptor WST-1, as a function of NADH concentration. At all NADH concentrations tested  Glu37 metabolized NADH at a significantly faster rate than Asp37. The recent discovery of renalase has elucidated a novel pathway for the homeostatic control of circulating catecholamines. Data from the International HapMap Consortium has identified a single common non-synonymous single-nucleotide polymorphism in the human renalase gene that results in an aspartic acid to glutamic acid substitution at codon 37 (Glu37Asp) in the flavin-adenine dinucleotide-binding site. In the present study of 590 persons with stable coronary artery disease, we report an association of CC (Asp/Asp) homozygosity at codon 37 with cardiac hypertrophy, dysfunction, and ischemia. Although this is a conservative substitution (Glu37Asp), it does occur in a critical region of the protein, namely the FAD binding domain, And it is, therefore, not surprising that compared to Glu37, Asp37 has a significantly lower affinity for NADH and a reduced maximal velocity. These findings raise the intriguing possibility that decreased activity of the enzyme renalase may contribute to cardiovascular disease.in vivo relevance of renalase in humans are lacking. In a large case-control study of a northern Han Chinese population, Zhao et al found an association of the (Glu37Asp) polymorphism with essential hypertension  At baseline, renalase activity in blood mirrors sympathetic tone 2). This supports a mechanistic link between the renalase-catecholamine axis and left ventricular hypertrophy, and extends this finding to humans. Further studies in animal models are warranted to investigate the relative contributions of renalase secreted from the kidney and the heart to the inhibition of cardiac hypertrophy.Accumulating evidence implicates excessive circulating catecholamines in the pathogenesis of left ventricular hypertrophy, a powerful independent risk factor for cardiovascular death and morbidity The association of the Glu37Asp polymorphism with cardiac dysfunction has not previously been reported. Renalase infusion in rats results in an increase in stroke volume, which may reflect a compensatory response to decreased heart rate. However, since renalase also reduces cardiac contractility, an alternative explanation is that renalase-induced metabolism of circulating catecholamines increases cardiac lusitropy and optimizes diastolic-systolic coupling. We found essentially similar odds ratios for both systolic and diastolic dysfunction associated with Asp/Asp homozygosity in multivariable adjusted models further suggesting an influence of the renalase-catecholamine axis on both aspects of cardiac function. Detailed hemodynamic studies examining the effects of renalase administration on diastolic function would be of interest in this regard.Cardiac ischemia, whether acute or chronic, is associated with a significant elevation in circulating catecholamines that promote and exacerbate myocardial infarction a priori selection of the only known common coding polymorphism in renalase, the genotype-phenotype associations reported are biologically plausible and unlikely to represent false positive associations The main strength of the present study is the collection of detailed phenotypic data derived from comprehensive resting and stress echocardiography in all participants. Combined with the In conclusion, we found an association of the renalase Glu37Asp polymorphism  with cardiac hypertrophy, dysfunction, and ischemia in a cross-sectional study of individuals with stable coronary artery disease and normal renal function. The renalase pathway is a novel regulator of circulating catecholamine levels. Future studies should be aimed at determining whether there is a potential therapeutic role for renalase replacement in persons at high risk of cardiovascular morbidity and mortality.


Constrictive pericarditis can lead to paradoxical interventricular septal motion. Displacement encoding with stimulated echoes (DENSE) magnetic resonance imaging (MRI) provides a method for quantifying myocardial motion and strain. A case of constrictive pericarditis is presented and the diastolic ‘septal bounce’ is clearly evident in both anatomical and DENSE ciné MRI images. (See video link to full-text electronic article). The postoperative systolic septal wall-motion abnormality of cardiac surgery is portrayed with greater precision by DENSE than anatomical ciné MRI images. Constrictive pericarditis is present when a fibrotic, thickened, and adherent pericardium restricts diastolic filling of the heart. In early diastole, when intracardiac volume is less than that defined by the stiff pericardium, diastolic filling is unimpeded, and early diastolic filling occurs abnormally rapidly because venous pressure is elevated. The rapid early diastolic filling which is halted abruptly when intracardiac volume reaches the limit set by the non-compliant pericardium is reflected by the abrupt displacement of the interventricular septum into the left ventricle (LV) during early diastole .2Mycobacterium tuberculosis after 16 days of culture, thus confirming a diagnosis of tuberculous pericarditis. He was commenced on standard anti-tubercular medication for six months and a tapering course of adjunctive oral prednisone for six weeks. He did not respond as expected to this treatment, such that on 3 March 2006 he developed clinical and echocardiographic features of effusive constrictive pericarditis. Pericardiectomy was performed on 20 July 2006. Post-operative examination in September 2006, however, demonstrated clinical features of persistent constrictive pericarditis and no bundle branch block on the electrocardiogram.A 47-year-old man presented on 6 January 2006 with signs and symptoms of pericardial effusion. Pericardiocentesis yielded 550 ml of straw-coloured fluid, which grew Magnetic resonance imaging (MRI) was performed on 5 October 2006, showing the diastolic ‘septal bounce’ of constriction on steady-state free precession (SSFP) imaging in a midventricular short-axis view [DENSEThe ciné DENSE motion trajectory positions at four cardiac phases from a mid-ventricular short-axis slice are shown in 6The patient was treated conservatively with anti-tubercular medication and diuretics, and his signs and symptoms gradually improved without further surgical intervention. Ciné DENSE is shown to define the dominant postoperative systolic septal wall motion abnormality of cardiac surgery with greater precision than anatomical ciné MRI images.


Trans-mitral Doppler produces complex patterns with a great deal of variability. There are several confusing numerical measures and indices to study these patterns. However trans-mitral Doppler produces readymade data visualization by pattern generation which could be interpreted by pattern analysis. By following a systematic approach we could create an order and use this tool to study cardiac function.In this new approach we eliminate the variables and apply pattern recognition as the main criterion of study. Proper terminologies are also devised to avoid confusion. In this way we can get some meaningful information.vis-à-vis the Starling's Law applied to the left atrium.Trans-mitral Doppler should be seen as patterns rather than the amplitude. The hypothesis can be proven by logical deduction, extrapolation and elimination of variables. Trans-mitral flow is also analyzed Trans-mitral Doppler patterns are not just useful for evaluating diastolic function. They are also useful to evaluate systolic function. By following this schema we could get useful diagnostic information and therapeutic options using simple pattern recognition with minimal measurements. This simplified but practical approach will be useful in day to day clinical practice and help in understanding cardiac function better. This will also standardize research and improve communication. Echocardiography has become an important diagnostic tool in clinical cardiology. With the advent of Doppler studies, analysis of trans-mitral left ventricular Doppler filling patterns (LV DFP) has assumed importance. LV DFPs When the scientific world is betting on the power of human visual intelligence to find patterns in data, it is unfortunate that these readymade transmitral Doppler patterns are not used as such. Instead the present methodologies reconvert these useful patterns to numerical data. The value and extraordinary power of data visualization for analysis is quickly gaining recognition. This should also be used in the analysis of Doppler patterns.Trans-mitral Doppler interrogation produces a wide range of patterns. But the predominant feature is a biphasic flow. The early flow is the E wave and the late flow is the A wave. Occasionally a third wave may be noted between the two. This coincides with the partial opening of the mitral valve following the M-mode F point. So this wave may be termed G wave. In this discussion we are only considering flow Doppler patterns. The author is of the view that there are methodological flaws in tissue Doppler. Hence thThe highly variable nature of these patterns makes them difficult for linear analysis. In this new approach we eliminate the variables and apply pattern recognition as the main criterion of study. Proper terminologies are also devised to avoid confusion. As a first step in this elimination process, this method is used in normal valves only. By normal valves we also exclude valve stiffness, which could affect Doppler patterns. An example is mitral valve stiffness not amounting to stenosis.There is a common understanding that trans-mitral Doppler patterns are used to study diastolic function. However, these patterns should be considered as a 'tool' which just happens to occur in diastole. Then we can have a wider perspective and use the 'tool' to study systolic function too. Hence these patterns are called 'filling patterns' without reference to the cardiac cycle. The inferences derived from the Doppler pattern depend on the systolic function. It is like using the measuring tape to measure abdominal girth in a female. The inference from the measurement will depend on whether the patient is pregnant. It is a case of same tool, similar measurement but different inference depending on the clinical situation.The various numerical indices used to study these patterns have been complex and confusing for routine clinical practice. All these indices are dependant on several variables like chamber properties, pressure changes, heart rate etc. However by pattern recognition these can be simplified so that useful information can be derived in day-to-day practice. Thus 2 types of information can be derived from LV DFP. 1. Chamber properties 2. Hemodynamic information.LV DFP can be used to study the chamber properties. This is more relevant when the systolic function is normal (see below). Here 2 recognizable patterns are discernable. 1. Relaxation abnormality pattern or 'R pattern' 2. Compliance abnormality pattern or 'C pattern'. Many indices are used for chamber properties. However the commonly used index is E wave deceleration time. There are practical problems in determining the deceleration time. For example with the available software it is difficult to accurately determine the deceleration time in a curved "ski-slope" Doppler tracing. A simpler and more accurate method would be determination of the early filling time (EFT). This is the duration of the E wave from the beginning of the E wave to the end . Here LV DFP could have diagnostic roles. In these cases of 'pure diastolic dysfunction' the 'impaired relaxation' or R pattern could point to hypertension, diabetes, coronary artery disease or hypertrophic cardiomyopathy. It could also signify age related changes. The 'comsystolic dysfunction rather than severity of diastolic dysfunction (see below). By impaired systolic function we could also include cases with wall motion abnormalities with apparently normal ejection fraction and apparently normal overall LV contractility. Pattern 1 shows a small E wave and a large A wave could indicate mild hemodynamic impairment. Pattern 2, shows a 'pseudo-normalization' pattern denoted by an apparently normal E and A wave. Pattern 2 could indicate moderate impairment. Finally, pattern 3 is a very prominent E wave and a miniscule A wave. Pattern 3 could indicate severe hemodynamic impairment and is associated with the worst prognosis.[In cases with impaired systolic function, it is primarily study of systolic function ('systology'). Systolic dysfunction is usually associated with increased LV end diastolic pressure and left atrial pressure. This canrognosis. In casesrognosis. In seriaDiseases with combined systolic dysfunction and abnormal chamber properties will show a combination of the above features. For example, in a case of systolic dysfunction with a pattern 1.5 if there is prolonged EFT then the pattern could be described as 1.5 R.any cardiac disease can cause LV diastolic dysfunction to variable extents  will compensate for the atrial output. The progression of the A wave reflects the Starlings Law , a C pattern? , or a combination RC pattern? The R pattern could be age related and non-cardiac pathologies like obesity, sleep apnoea, deconditioning or renal failure could produce patterns between 0 and 1.LV DFP can only be interpreted in the context of systolic function.If the systolic function is not satisfactory, then one can assess the severity and prognosticate using the LV DFP as described above. In cases with wall motion abnormality with apparently normal LV contractility a pattern to the right of 0 could signify early systolic dysfunction. Remember, In a complex situation, it is possible to identify the problem by recognizing the predominant hemodynamic pattern and factoring in the complicating chamber property.The accuracy of interpretation of LV DFP is best in the young (<50 years) where age related changes due to atrial, ventricular and valvular stiffness are eliminated.A clinical example will demonstrate the use of this methodology. Consider a 35 year old patient with myocardial infarction. There was apparently acceptable eye-ball LV function with one akinetic apical segment. The trans-mitral Doppler showed E<A with a normal EFT. This is pattern 1 and could signify a predominantly mild LV systolic dysfunction rather than 'diastolic dysfunction'. A practical example of pattern progression in disease state is shown in figure systolic dysfunction and should make the clinician search diligently for wall motion abnormalities. DFP could also indicate prognosis.[Transmitral Doppler patterns are not just useful for evaluating diastolic function. They are also useful to evaluate systolic function. By following this schema, it will be possible to fit these patterns into specific groups thus aiding in diagnosis and treatment. The detection of R pattern should make the clinician search aggressively for causes like hypertension, diabetes, ischaemic heart disease and cardiomyopathy in the young (below 50 years of age). Similarly, the detection of pattern 1 in a young individual could also indicate rognosis.,22Doppler patterns are easy to acquire and are intriguing. However the interpretation is difficult. This methodology presents a gross simplification of a complex phenomenon. But by following this systematic analysis we could get useful diagnostic information and therapeutic options. This methodology uses pattern recognition with minimal measurements. The only measurement of EFT can also be done away with once the operator is used to the display sweep settings. It is practical and can be applied in day to day clinical practice. The precise terms will improve communication and standardize research.LV: Left ventricle; DFP: Doppler filling pattern; R-pattern: Relaxation abnormality pattern; C-pattern: Compliance abnormality pattern; RC-pattern: Combination of R and C patterns; EFT: Early filling time; LAP: Left atrial pressure.The author declares that they have no competing interests.


Cardiac time intervals have been described as a measure of cardiac performance, where prolongation, shortening and delay of the different time intervals have been evaluated as markers of cardiac dysfunction. A relatively recently developed method with improved ability to measure cardiac events is Tissue Doppler Imaging (TDI), allowing accurate measurement of myocardial movements.We propose the state diagram of the heart as a new visualization tool for cardiac time intervals, presenting comparative, normalized data of systolic and diastolic performance, providing a more complete overview of cardiac function. This study aimed to test the feasibility of the state diagram method by presenting examples demonstrating its potential use in the clinical setting and by performing a clinical study, which included a comparison of the state diagram method with established echocardiography methods . The population in the clinical study consisted of seven patients with non ST-elevation myocardial infarction (NSTEMI) and seven control subjects, individually matched according to age and gender. The state diagram of the heart was generated from TDI curves from seven positions in the myocardium, visualizing the inter- and intraventricular function of the heart by displaying the cardiac phases.The clinical examples demonstrated that the state diagram allows for an intuitive visualization of pathological patterns as ischemia and dyssynchrony. Further, significant differences in percentage duration between the control group and the NSTEMI group were found in eight of the totally twenty phases (10 phases for each ventricle), e.g. in the transition phases (Pre-Ejection and Post-Ejection). These phases were significantly longer (> 2.18%) for the NSTEMI group than for the control group (p < 0.05). No significant differences between the groups were found for the established echocardiography methods.The test results clearly indicate that the state diagram has potential to be an efficient tool for visualization of cardiac dysfunction and for detection of NSTEMI. Cardiac time intervals have been described as a measure of cardiac performance and as a tool to help understand and illustrate the mechanics of the heart. Prolongation, shortening and delay of the different time intervals have been evaluated as markers of cardiac dysfunction ,2. MethoWe propose the state diagram of the heart as a new visualization tool for cardiac time intervals, presenting comparative, normalized data of systolic and diastolic performance, providing a more complete overview of cardiac function . This toState diagrams are used to describe the behaviour of systems, often applicable in technical systems in order to explain and simplify the way the system operates. This study is the first attempt, in a comprehensive work, to present the function of the right and left side of the heart using state diagrams. The work has, in particular, focused on rational ways to find the mechanical criteria for how the heart goes from one state to the next, which defines the regional and global function of the heart. The duration of the different cardiac states, heart phases, are presented in the state diagram. The primary aim of the study was to test the feasibility of the state diagram method by performing a clinical study, which included a comparison of the state diagram method with established echocardiography methods, and by presenting clinical examples demonstrating its potential use in the clinical setting.The state diagrams were generated from myocardial velocity curves recorded by TDI. Image acquisition was performed in the left lateral decubitus position using a Vingmed System Vivid 7  with a standard 2D transducer (M3S) or a 3D matrix transducer (3V) for triplane imaging. Images were obtained in three imaging planes: apical long-axis (APLAX), apical four-chamber (4CH) and apical two-chamber (2CH) and were imported to EchoPAC Dimension . In the two-dimensional gray scale image with superimposed colour Doppler, a circular region of interest (ROI) with a diameter of 4 mm was positioned in the basal segment of the antero-septal, anterior, antero-lateral, infero-lateral, inferior, infero-septal (left ventricle) and medial wall . The mean velocity curve for each ROI, in total seven, was stored digitally in a format compatible with Matlab R2006b, where the software was developed. The study has been carried out in accordance to the declaration of Helsinki and ethical approval was obtained from the local ethics committee. All participants gave informed consent.The generation of a state diagram was initiated with a detection of triggering points in each tissue velocity curve. The cardiac cycle was divided into six main phases in the tissue velocity curve: Pre-Ejection, Ventricular Ejection, Post-Ejection, Rapid Filling, Slow Filling and Atrial Contraction. Ventricular Ejection was subdivided into an early, a mid and a late phase whereas the Atrial Contraction and the Rapid Filling were subdivided into two phases: a early phase and a late phase. Figure Pre-Ejection was defined as the time interval between the first zero-crossing in the tissue velocity curve after the R-wave in the ECG-curve and the zero-crossing after the typical biphasic pattern with a negative and a positive velocity spike, which are involved in the closure of the inlet valves. This occurs at the ascending limb of the tissue velocity curve at the beginning of Ventricular Ejection, see Figure Ventricular Ejection was defined as the time interval between the end of Pre-Ejection and of the start of the Post-Ejection phase. Early Ventricular Ejection was defined as the first acceleration phase lasting until the largest derivative change in the ascending limb of the tissue velocity curve. Late Ventricular Ejection was defined as starting with the largest derivative change in the descending limb of the tissue velocity curve. The time between early and late Ventricular Ejection was called mid Ventricular Ejection.Post Ejection was defined as the time interval between the zero-crossing of the descending limb of the systolic tissue velocity curve and the zero-crossing after the biphasic movements with a negative and a positive velocity spike, which are involved in the closing procedure of the outlet valves, see Figure Rapid Filling was defined as the time between the endpoint of Post-Ejection and the time point when the velocity derivative shifts from a positive value to a value close to zero. The phase was divided into an early and a late part by the minimum velocity value during this phase.Slow Filling was defined as the time from the end of Rapid Filling until the start of Atrial Contraction.Atrial Contraction was defined as starting with the largest decline in the tissue velocity curve occurring in connection to the P-wave in the ECG. The end point equalled the start of the Pre-Ejection phase for the next cardiac cycle. The Atrial Contraction was divided into an early and a late phase with the minimum velocity value as the dividing event.The outermost circular segment in the state diagram corresponds to the antero-septal wall, followed by the anterior, antero-lateral, infero-lateral, inferior, infero-septum and medial wall, see Figure State diagrams were generated at rest in four typical cases, the healthy subject, the athlete, the ischemic subject and the dyssynchronic subject. Figure A sample of seven patients with NSTEMI was randomly selected from the echo archive at Karolinska University Hospital in Huddinge, Sweden. The control group was individually matched according to age and gender, with no symptoms of ischemic heart disease at the time for the investigation. Each group consisted of 6 men and 1 woman. The mean age was 58.2 (SD 7.6) in the diseased group and 58.7 (SD 8.0) in the control group and the mean heart rate was 61.0 (SD 5.7) and 62.4 (SD 6.6), respectively. The characteristics of the studied subjects are described in Table Conventional two-dimensional echocardiography and colour TDI images were recorded for both groups. Images were obtained at rest as described above with a standard 2D transducer (M3S) with a frame rate of the TDI data varying between 100 and 150 frames/s. The flow velocity (E) and the mean tissue velocity (E') of the early diastolic inflow in the septal and lateral corner of the mitral annulus were used for calculation of the E/E' ratio. Left ventricular Ejection Fraction (LVEF) was measured with the biplane Simpson's method and wall motion score index (WMSI) was performed by an experienced stress echo reader. State diagrams for each subject were generated with the software developed in Matlab, see above for more details.All statistical analysis was performed using standard statistical software (SPSS version 16.0). For the individually matched couples, a paired t-test was performed to test the difference in percentage duration of each phase in the global state diagram (control-NSTEMI). A total of twenty phases were tested, ten in the left ventricle and ten in the right ventricle. A paired t-test was also used to test the difference in E/E' ratio, LVEF and WMSI between the groups.For the totally twenty phases in the state diagram, significant differences in percentage duration between the control group and the NSTEMI group were found in eight. When the values were positive, the percentage duration was longer for the control group than for the NSTEMI group. In the late Atrial Contraction, significant differences were found for both ventricles, 2.27% SD 1.5 (p = 0.008) for the left ventricle and 2.40% SD 1.6 (p = 0.007) for the right ventricle. The difference in percentage duration of Pre-Ejection was significant in both ventricles, -3.52% SD 1.5 (p = 0.001) and -2.18% SD 2.2 (p = 0.041) for the left and the right ventricle, respectively. The difference in percentage duration for mid Ventricular Ejection in the right ventricle was 2.09% SD 2.1 (p = 0.038) and for Post-Ejection, -2.70% SD 2.5 (p = 0.029) for the left and -2.94% SD 1.5 (p = 0.002) for the right ventricle. For early Rapid Filling in the left ventricle the difference in percentage duration was 1.65% SD 1.5 (p = 0.026).The results from the statistical analysis regarding the state diagram variables are shown in Table Table This study was an attempt to test the clinical feasibility of the state diagram as a visualization tool for cardiac mechanics. The state diagram was designed to present the mechanics of both the left and the right side of the heart since they each have an AV-piston. The myocardium is the power source for the pumping function. It has a flexible structure with a constant volume, which means that generation and release of tension forces within the myocardium can occur with low or no external volume changes, i.e. changes of the epicardial borders . The myoThe phases in the state diagram have been defined in accordance with the DDP-principle in order to illustrate the mechanics of the heart, and henceforth the heart will be described as a DDP. Since the motion of the AV-pistons largely represents the mechanics of the heart, the myocardial velocities were chosen to be measured in the basal segments of the heart, near the AV-pistons. The crucial task for the heart is to preserve flow and muscle dynamics throughout the cardiac cycle and to keep a constant inflow to the heart when the AV-pistons have to be accelerated, retarded and change direction. The more or less constant inflow to the heart is achieved by suction of blood into the atria during Ventricular Ejection and continuous need of further inflow during the hydraulic return of the AV-piston during Rapid and Slow Filling.Motion shifts of the piston associated with opening and closing of the valves are critical events that affect the performance of the heart. These events have in this study been included in the phases Pre-Ejection and Post-Ejection, names which refer to their close link to the beginning and ending of Ventricular Ejection. Pre-Ejection and Post-Ejection differ from the traditionally defined isovolumic phases by including volume redistributions between the atria, the ventricles and its outlets. During Atrial Contraction, the AV-pistons are drawn towards the inlet areas of the atria, the base of the heart, whereas during Pre-Ejection they are drawn towards the apex of the heart ,18. ThisEvents connected to Pre-Ejection and Post-Ejection are considered to result from pre- and postsystolic reshaping of the muscles in the ventricles . Pre-EjeThe results of this study show that the state diagram has potential to be used for visualization of various cardiac dysfunctions. It also demonstrates the state diagram to be a more sensitive tool for detection of NSTEMI compared to other established echocardiographic variables such as E/E' ratio, LVEF and WMSI, since no significant differences were found in these variables. Moreover, it must be considered that the state diagram method had the ability to separate the groups, even though the control group demonstrated some clinical features. The novelty with this method is the visualization by presenting a clear overview of cardiac mechanics according to the DDP-principle. With this approach it is possible to create a state diagram, which allows for evaluation of parallel information by displaying the relationships between different phases in the cardiac cycle.Though the state diagram method is based on TDI, its accuracy and reproducibility can be compared with the accuracy and the reproducibility established for TDI. TDI has been validated and a reasonably good reproducibility has been found ,21. HoweThe pre- and postsystolic phases, called Pre-Ejection and Post-Ejection in this study, are important considerations when evaluating cardiac function and the prolongation of these time intervals are associated with systolic and diastolic ventricular dysfunction -24. The Furthermore, the clinical examples of the ischemic and the dyssynchronic subjects indicate that the state diagram can detect and display regional heart muscle disturbances such as infarcted and ischemic movement patterns and dyskinetic functions of the muscle cells. Ventricular dyssynchrony disturbs the synchronous pumping and relaxation function of the heart leading to a reduced diastolic filling time and a post-systolic regional contraction , two facCare must be taken when interpreting the results since the number of patients included in the study was small. The state diagrams for the patients in the clinical study and for the ischemic clinical example were generated using a 2D transducer. A 2D transducer renders the state diagrams dependent on recording conditions since the timing, based on velocities from different cardiac cycles, is displayed simultaneously. This is negligible in most cases since the beat-to-beat differences during an examination are small. In addition, the differences in heart rate between each NSTEMI patient and the matched control might have affected the results of the clinical study. However, these differences were not systematic and had most likely only a moderate influence on the results.The test results clearly indicate that the state diagram is feasible in the clinical setting and has potential to be an efficient tool for visualization of cardiac dysfunction and for use in the detection of NSTEMI. However, larger population studies are needed to further test the feasibility of state diagrams in this regard.Potential conflicts of interest are as follows: JJ is on the board in GrippingHeart AB and his Ph.D work is partly founded by GrippingHeart AB. SL partly owns GrippingHeart AB.ML, AB, JJ, SL and LÅB participated in contributions to conception, analysis and interpretation of data. ML, AB and JJ contributed with the programming work. ML and AB wrote the manuscript, critically reviewed by JJ and SL. RW was the supervisor of echo examinations and reviewed the manuscript for relevant intellectual content. All authors read and approved the final manuscript.


Prostacyclin inhalation is increasingly used to treat acute pulmonary hypertension and right ventricular failure, although its pharmacodynamic properties remain controversial. Prostacyclins not only affect vasomotor tone but may also have cAMP-mediated positive inotropic effects and modulate autonomic nervous system tone. We studied the role of these different mechanisms in the overall haemodynamic effects produced by iloprost inhalation in an experimental model of acute pulmonary hypertension.In this prospective, randomized, placebo-controlled animal study, twenty-six pigs (mean weight 35 ± 2 kg) were instrumented with biventricular conductance catheters, a pulmonary artery flow probe and a high-fidelity pulmonary artery pressure catheter. The effects of inhaled iloprost (50 μg) were studied in the following groups: animals with acute hypoxia-induced pulmonary hypertension, and healthy animals with and without blockade of the autonomic nervous system.P = 0.0013), a selective reduction in right ventricular afterload  and a significant increase in left ventricular end-diastolic volume . Interestingly, right ventricular contractility was reduced after iloprost-treatment , whereas ventriculo-vascular coupling remained essentially preserved . In healthy animals, inhaled iloprost had only minimal haemodynamic effects and produced no direct effects on myocardial contractility, even after pharmacological blockade of the autonomic nervous system.During pulmonary hypertension, inhalation of iloprost resulted in a 51% increase in cardiac output compared with placebo (5.6 ± 0.7 versus 3.7 ± 0.8 l/minute; In animals with acute pulmonary hypertension, inhaled iloprost improved global haemodynamics primarily via selective pulmonary vasodilatation and restoration of left ventricular preload. The reduction in right ventricular afterload is associated with a paradoxical decrease in right ventricular contractility. Our data suggest that this reflects an indirect mechanism by which ventriculo-vascular coupling is maintained at the lowest possible energetic cost. We found no evidence for a direct negative inotropic effect of iloprost. Because of its marked pulmonary vasodilating effects, ease of administration and lack of toxicity, intermittent nebulization of iloprost has become an established therapy in chronic pulmonary hypertension (PHT)  and is i2 [PGI2]), improves cardiac performance in PHT. PGI2 stimulates the intracellular synthesis of cAMP [2 infusion in various models. A clinical study demonstrated that in PHT, inhaled iloprost increases cardiac output more than nitric oxide [There is evidence that ventricular afterload reduction may not be the sole mechanism by which iloprost, the stable carbacyclin derivative of prostacyclin  [This investigation conforms to the ed 1996)  and was Twenty-six pigs (mean weight 35 ± 2 kg) were included in the study. After intramuscular premedication with ketamine (20 mg/kg), piritramide (1 mg/kg) and atropine (0.5 mg), anaesthesia was induced with intravenous sodium pentobarbital (12 mg/kg). After endotracheal intubation, anaesthesia was maintained with a continuous intravenous infusion of sodium pentobarbital (3 to 4 mg/kg per hour), sufentanil (3 μg/kg per hour) and pancuronium (0.2 mg/kg per hour). Mechanical ventilation with a mixture of oxygen and room air was adjusted to achieve normocapnia and normoxia, as controlled with arterial blood gas measurements taken at regular intervals . A balanced electrolyte solution was administered at a rate of 8 ml/kg per hour. Normothermia was maintained during the entire procedure using an infrared heating lamp.A 7.5-Fr central venous catheter was inserted into the femoral vein. A 16-G arterial catheter was advanced into the descending aorta via the femoral artery. A lateral cut-down was performed in the cervical region and an 8.5-Fr introducer sheath was inserted into the left carotid artery.Via a midline sternotomy, a tourniquet was placed around the inferior vena cava (IVC) for controlled reductions in ventricular preload. A 20 mm nonrestricting perivascular flow probe  was placed around the main pulmonary artery (PA). A 6-Fr micro-tipped pressure transducer  was advanced into the PA via a stab wound in the pulmonary outflow tract with its tip just distal to the flow probe. Combined micro-tip multisegment pressure-volume catheters  were inserted into the right ventricle and left ventricle, through a stab wound in the pulmonary outflow tract and via the left carotid artery, respectively. Correct position of the conductance catheters was confirmed with radiography.Haemodynamic measurements were started after completion of instrumentation and 30 minutes of haemodynamic stabilization. Measurements were always performed with the ventilation suspended at end-expiration. Data were acquired during steady-state conditions and during a brief period of IVC occlusion to obtain a series of successive heart beats at progressively lower end-diastolic volumes, for the calculation of contractile indices and pulmonary pressure-flow relationships.In 16 animals acute PHT was induced with hypoxic pulmonary vasoconstriction. After control haemodynamic measurements, nitrogen was added to the inspiratory gas mixture and the fraction of inspired oxygen reduced until the mean PA pressures exhibited an increase of at least 50% compared with baseline values  were examined with (n = 5) and without (n = 5) blockade of the ANS. Blockade of the ANS was accomplished with atropine methyl nitrate (3 mg/kg), propranolol hydrochloride (2 mg/kg) and hexamethonium bromide .In a subsequent study, including 10 pigs, the effects of inhaled iloprost  connected to the inspiratory limb of the ventilator circuit. This nebulizer is characterized by a mass median aerodynamic diameter of 2.3 μm and a geometric standard deviation of 1.6. Nebulization times were not prefixed as in the chosen mode of delivery; the device stops automatically when aerosolization of the volume filled into the nebulizer is completed. Mean nebulization times in our experimental setting were 617 ± 67 seconds. After stopping iloprost/placebo inhalation, an average fluid amount of 0.50 ± 0.12 ml was found to remain in the nebulizer.Both iloprost and the placebo solution were aerosolized using a commercially available ultrasonic nebulizer , in one of which the excitation frequency had been adjusted from 20 to 19 kHz in order to avoid cross-talk . The the® .All parameters were digitized at 333 Hz and stored for off-line analysis with custom-made algorithms written in MatlabAs previously described, ventricular contractility was quantified with the slope of the end-systolic pressure-volume relationship . Ventriculo-vascular coupling was described as the ratio of Emax over Ea . PA comp© for Windows, version 6.0; Statsoft, Tulsa, OK, USA). To test the global hypothesis that iloprost has an effect on haemodynamic variables in PHT, the group versus time interaction was analyzed using repeated measures analysis of variance with the within-factor time and the grouping factor treatment (iloprost versus control). Likewise, the effects of ANS blockade were tested with the grouping factor autonomic blockade versus no ANS blockade [t-test, respectively. The Bonferroni-Holm adjustment was used to correct for multiple comparisons [The sample size was calculated based on previous experiments. Power analysis revealed a minimal sample size of five pigs to detect a 33% effect in mean PA pressure and RV-Mw, when a level of significance of 0.05 and a power of 80% were to be achieved. Results were statistically analyzed using a commercially available software package  and parallel (disturbance of diastolic and systolic LV function by leftward shifting of the septum) ventricular interaction ,44. In fSeveral limitations of the present study should be acknowledged.Data were obtained in an open chest-open pericardium model using general anaesthesia. These experimental conditions may significantly affect cardiovascular mechanics, but we considered them relevant to the setting of cardiac surgery, in which RV dysfunction is an important risk factor for perioperative mortality . In addiRV coronary perfusion pressure was calculated rather than measured directly. Because no consensus exists on this matter, we opted to use the difference between systolic arterial pressure and RV systolic pressure, taking into consideration the fact that an important part of right coronary artery flow occurs during systole and that, in PHT, RV coronary artery flow is impaired proportionally to RV systolic pressures .2 might not be related simply to vasodilatation but also to other mechanisms involving pulmonary vascular remodeling [Finally, the influence of ANS blockade on the effects of iloprost was not studied in PHT. In pilot experiments, however, ANS blockade in hypoxia-induced PHT was associated with lethal cardiovascular collapse, highlighting the importance of the intact sympathetic nervous system, as shown recently in our laboratory . Moreovemodeling . In any 2 [In animals with acute PHT, inhalation of iloprost resulted in selective pulmonary vasodilation, which – in contrast to previous findings with systemic application of PGI2  – was as• In acute PHT inhaled iloprost improves general haemodynamics, primarily through selective pulmonary vasodilatation.• Inhaled iloprost effectively restores LV preload via the mechanism of ventricular interdependence.• Inhaled iloprost indirectly decreases RV contractility but preserves right ventriculo-vascular coupling at lower oxygen cost.2: prostaglandin I2; PHT: pulmonary hypertension; PQ: pressure-flow; PVA: pressure-volume area; PVR: pulmonary vascular resistance; RPP: rate-pressure product; RV: right ventricular.ANS: autonomic nervous system; Ea: effective arterial elastance; Emax: slope of the end-systolic pressure-volume relationship; IVC: inferior vena cava; LV: left ventricular; Mw: slope of the preload-recruitable stroke work relationship; PA: pulmonary artery; PGIIn the past five years, SR and WB have received honoraria for lectures from the manufacturer of iloprost. From 2003 to 2004, the Department of Anaesthesiology of the University of Aachen held a research grant from Schering AG, the former manufacturer of iloprost.SR conceived of the study and – together with CM – designed the study, carried out the animal experiments, performed the data acquisition and the statistical analysis, and wrote the manuscript. PC wrote the software algorithms for the data analysis, participated in the statistical analysis and helped to draft the manuscript. WB participated in the study design and coordination. PW is responsible for the final study design, participated in the animal experiments, supported the data acquisition and the statistical analysis, and edited the final manuscript. All authors read and approved the final manuscript.Additional file Click here for fileAdditional file Click here for fileAdditional file Click here for fileAdditional file Click here for fileAdditional file Click here for fileAdditional file Click here for fileAdditional file Click here for file


Standard extracellular cardiovascular magnetic resonance (CMR) contrast agents (CA) do not provide differentiation between acute and older myocardial infarcts (MI). The purpose of this study was to develop a method for differentiation between acute and older myocardial infarct using myocardial late-enhancement (LE) CMR by a new, low molecular weight contrast agent.Dogs (n = 6) were studied in a closed-chest, reperfused, double myocardial infarct model. Myocardial infarcts were generated by occluding the Left Anterior Descending (LAD) coronary artery with an angioplasty balloon for 180 min, and four weeks later occluding the Left Circumflex (LCx) coronary artery for 180 min. LE images were obtained on day 3 and day 4 after second myocardial infarct, using Gd(DTPA) (standard extracellular contrast agent) and Gd(ABE-DTTA) , respectively. Triphenyltetrazolium chloride (TTC) histomorphometry validated existence and location of infarcts. Hematoxylin-eosin and Masson's trichrome staining provided histologic evaluation of infarcts.Gd(ABE-DTTA) or Gd(DTPA) highlighted the acute infarct, whereas the four-week old infarct was visualized by Gd(DTPA), but not by Gd(ABE-DTTA). With Gd(ABE-DTTA), the mean ± SD signal intensity enhancement (SIE) was 366 ± 166% and 24 ± 59% in the acute infarct and the four-week old infarct, respectively (P < 0.05). The latter did not differ significantly from signal intensity in healthy myocardium (P = NS). Gd(DTPA) produced signal intensity enhancements which were similar in acute (431 ± 124%) and four-week old infarcts , and not statistically different from the Gd(ABE-DTTA)-induced SIE in acute infarct. The existence and localization of both infarcts were confirmed by triphenyltetrazolium chloride (TTC). Histologic evaluation demonstrated coagulation necrosis, inflammation, and multiple foci of calcification in the four day old infarct, while the late subacute infarct showed granulation tissue and early collagen deposition.Late enhancement CMR with separate administrations of standard extracellular contrast agent, Gd(DTPA), and the new low molecular weight contrast agent, Gd(ABE-DTTA), differentiates between acute and late subacute infarct in a reperfused, double infarct, canine model. Reinfarction occurs in 7-8% of cardiac patients with previous MI ,2. In a We have developed a family of CAs for CMR diagnosis of ischemic heart disease (IHD) -9. AmongGd(ABE-DTTA) has been successfully used for continuous detection of myocardial ischemia during 30 min of left anterior descending coronary artery (LAD) occlusion ,13. The In this study we have shown that Gd(ABE-DTTA) induces a LE effect in acute, but not in late subacute MI.Gd(ABE-DTTA) was synthesized, and samples were prepared, as described by Saab et al. . To guarAnimals were studied with a closed-chest, reperfused, double MI protocol described below. The smallest number of animals (n = 6) that still achieved statistical significance was used. MIs were generated in the LAD coronary artery territory and four weeks later in that of the left circumflex coronary artery (LCx) Figure .. To avost infarct) or the LCx (2nd infarct) and inflated for 180 minutes to create MI. Thereafter, the balloon was deflated to restore coronary circulation. Coronary angiography confirmed the reperfusion after balloon deflation.Animal protocol was approved by the University of Alabama at Birmingham IACUC in full compliance with the 'Guidelines for the Care and use for Laboratory Animals' (NIH). Six male hounds (18-20 kg) were used. Twelve hours prior to procedure food was taken away and 325 mg Aspirin given. Dogs were anesthetized with a Ketamine (5.0 mg/kg) and Diazepam(0.5 mg/kg) mixture, intubated, and connected to a Hallowell EMC Model 2000 respirator  operated with a tidal volume of 400 ml at a rate of 16 BPM. Anesthesia was maintained by continuous Isoflurane (2.5-3 volume %), and repeated Fentanyl (50-100 ug I.V. every 30 minutes), administration. Heart rate and blood oxygen saturation were monitored using a pulse-oxymeter placed on the animal's tongue. ECG electrodes were placed on the chest to record electrophysiological signs of myocardial ischemia and arrhythmias. The left femoral artery was separated surgically and an arterial sheath (6-8 French) was inserted. An I.V. line was placed to administer infusion and drugs. Heparin (100 IU/kg) was given intravenously to maintain the activated clotting time (ACT) above 300 seconds. A properly sized 2-3 mm angioplasty balloon was introduced under fluoroscopic guidance into the LAD  was dissolved in physiological saline to obtain 2% TTC solution. Slices were immersed in it at 37°C for 15 min and then rinsed with physiological saline. All TTC-stained slices were photographed with a high resolution digital camera. TTC-stained slices were used to validate the existence and location of infarcts.A 1.5T GE Signa-Horizon CV/i scanner  was used. A cardiac phased-array coil and ECG gating were employed. Breath-hold was performed at end-expiration. A 180°-prepared, segmented, inversion-recovery fast gradient-echo pulse was used with: Field of View (FOV) 30 cm, Echo Time (TE) 3.32 ms, Repetition Time (TR) two cardiac cycles (1100-1600 ms), slice thickness 10 mm. The Inversion Time (TI) was optimized to null the signal normal myocardium. Conventional cardiac angulation planes were set and short axis slices covering the entire left ventricle (LV) obtained (six slices per heart).In the first CMR session, a 0.2 mmol/kg Gd(DTPA)  bolus was administered intravenously. LE images were acquired 15-20 min thereafter. Gd(ABE-DTTA) was given intravenously at the end of the first CMR session. In the second CMR session, 24 h after Gd(ABE-DTTA) administration, LE images were similarly obtained.Post mortem tissue samples from the infarct and the peri-infarct regions were examined by histopathology. The samples were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm thickness. Hematoxylin-eosin and Masson's trichrome staining was performed.The existence of both acute and four-week old infarcts was validated and their anatomical localization determined by analyzing the TTC images. CMR Dicom images were imported as image sequences with the use of ImageJ . The endo- and epicardial contours of the LV muscle were traced manually and this circumscribed area was further analyzed. Based on the apicobasal localization and anatomical landmarks , CMR images acquired in the presence of the two different contrast agents, as well as the TTC slices, were matched. All CMR slices that contained a MI according to the corresponding TTC slices were categorized into four groups by anatomical localization of the infarct to either the four-week old or to the acute category, and by the contrast agent given.acute, Gd(DTPA)4 week, Gd(ABE-DTTA)acute, Gd(ABE-DTTA)4 week, to which two control groups, (Gd(DTPA)normal, Gd(ABE-DTTA)normal), i.e. normal myocardium with each of the two agents, have been added, bringing the number of data groups for analysis to six.To separate the acute and the four-week old infarcts for the analysis, the images of slices containing both types of MI were partitioned into two images, each reflecting one half of the tomographic slice. The partition was done, with ImageJ, along a straight line starting at the posterior interventricular groove (0° on the LV circumference), through the center point of the LV slice, ending at the 180° point on the LV circumference in the anterolateral region. Thus four groups of images were obtained for analysis: Gd(DTPA)To avoid observer bias, instead of manual contouring of the infarct and the healthy myocardial regions, a pixel-by-pixel analysis was performed. The pixel-by-pixel SI histogram of every CMR image, segmented in the above manner, was generated with ImageJ and these histograms were used for further analysis. First, the mean SI ± SD of healthy myocardium was determined by exporting the histograms to Origin 7.0 , and employing Gaussian curve fitting on each using the Levenberg-Marquardt algorithm . In agrei and SIn are the mean signal intensity in infarct and normal myocardium, respectively.where SIResults are reported as mean ± SD. Statistical analysis was carried out by SigmaStat . Two-way repeated measures analysis of variance was used to compare the SIE values among the six experimental groups. Normal distribution and equality of variances were tested. Although the test of normality failed, due to the equality of variances, the equality of group sizes, and the high power of the performed test (0.985 with α = 0.05), the assumption of the F test in the two-way ANOVA with repeated measures was not violated . Since aBoth the acute (LCx) and four-week old (LAD) infarcts were visible in Gd(DTPA)-enhanced LE images of all six dogs. The existence and localization of recent and four-week old infarct were confirmed by TTC. Histologic evaluation confirmed acute infarcts with coagulation necrosis, inflammation (mostly mononuclear), and multiple foci of calcification in the 4 days old infarct areas Figure .. HealinMean ± SD SIE values are shown in Table Gd(ABE-DTTA) was capable of differentiating between acute and four-week old infarcts as no LE effect was seen in the latter while one is clearly observable in the former. Acute MIs can be seen on the LE-CMR images enhanced with either Gd(DTPA) or Gd(ABE-DTTA). Older MIs are visible only by Gd(DTPA). Our general observations show in dogs that the agent's affinity to infarcted myocardial tissue disappears between days 10 and 14 following acute myocardial infarction. The question of the detailed kinetics of effect disappearance is currently under investigation.Saeed et al.  have rec1H MR and 23Na MR. Due to granulation tissue infiltrates and collagen deposition, the 23Na MR signal intensity in the MI area showed a significant decrease during infarct healing. Another approach by Abdel-Aty et al. [Other methods for the differentiation between new and longstanding MIs have also been published. In a recent publication Hillenbrand et al.  reportedy et al.  combinedy et al. . The spey et al.  used cony et al. ,5,25-27.The evolution of MI is a complex pathohistological process . SeveralOur study has some limitations. The LE images with the two different CAs were performed on two consecutive days. Thus, small differences in selection of the planes of the short-axis images between the two CMR sessions cannot be completely excluded. These differences, however, would not impair the outcome of our studies.On day 31, Gd(ABE-DTTA) was administered at the end of the first CMR session. It brings up the possibility, as Gd(DTPA)  might not have been fully cleared from the body yet, that the two contrast agents could potentially interact with each other. There is no chemical basis, however, for the assumption of interaction, and that it could have influenced the late enhancement phenomenon in the second CMR session, i.e. 24 hours later. Publications ,14 alrea201Thallium SPECT (Single Photon Emission Computed Tomography), where repeated delayed imaging is employed 3-4 h or 24 h following the administration of the radiotracer [Gd(ABE-DTTA) needs to be administered 24 hours before CMR imaging, and this introduces an inconvenience in a clinical setting. It may not be convenient (the cardiology ward and radiology are not necessarily close), nor practical for answering the urgent clinical question in a timely fashion. This mode of contrast agent administration, however, is not unknown in the practice of nuclear cardiology. For example, there is a protocol for the assessing of myocardial viability with rest redistribution iotracer .The slow clearance of Gd(ABE-DTTA) from the body suggests that the time available for undesired physiological effects, such as dissociation of Gd from the chelate, may be considerably longer than is the case for agents currently approved for human use. It is noteworthy, however, that Gd(ABE-DTTA)'s short- and long term physiological safety has been reported .In summary, we have shown that LE-CMR with separate administrations of Gd(DTPA) and Gd(ABE-DTTA) differentiates between acute and four-week old MIs in a reperfused, double infarct, canine model.Dr. Kirschner and Dr. Varga-Szemes are employees, Drs. A. Toth, L. Toth, P. Kiss, P. Suranyi and B. Ruzsics were employees, and Drs. A. Elgavish and G. Elgavish are officers of Elgavish Paramagnetics Inc.All authors participated in design and execution of experiments, analysis and interpretation of data, drafting and critical evaluation of manuscript. All authors have read and approved the manuscript.