Symptomatic gastrointestinal stromal tumours (GIST) are infrequent with an incidence of 12.7 per million inhabitants in the western population. We studied whether the incidence of GIST has further increased between 2003 and 2012 and assessed the frequency of mutations, risk groups, histological subtypes and immunohistochemistry results. From PALGA, the nationwide Dutch Pathology Registry, pathology excerpts from all patients with a GIST or GIST-like tumour between 2003 and 2012 were retrieved to calculate incidence rates. Full pathology reports were retrieved of resections in 2011 and 2012 to study the frequency of mutations, risk groups, histological subtypes and immunohistochemistry results. The incidence of GIST increased to 17.7 per million inhabitants in 2012 with a median age of 67 years. Mutational analysis was performed in 33.9% of patients with a resection between 2011 and 2012 . The percentage of high risk patients in the different risk classifications varied from 19.9% to 38.0% depending on the used classification. Only 35.9% of patients had diagnosis or revision of pathology diagnosis within three months in a designated GIST referral centre. No increase in proportion of central pathology reviews was found. Proportion of patients with mutational analysis increased over the years. The registered incidence of GIST, 17.7 per million inhabitants in 2012 in the Netherlands, is still rising. Despite incorporation in the ESMO GIST guidelines since 2008 for mutational testing and since 2010 for central review of pathology, both are performed in a minority of patients.The online version of this article (10.1007/s00428-017-2285-x) contains supplementary material, which is available to authorized users. The most common mesenchymal tumours of the gastrointestinal tract are the gastrointestinal stromal tumours (GISTs) . ClinicaPrimary treatment remains surgery and when non-resectable, imatinib has considerably improved prognosis of these patients , 12–15. The diagnosis of GIST is based on morphology and CD117 and/or DOG1 immunohistochemistry , 25, 26.In 2004, a nationwide survey was performed in the Netherlands to estimate the incidence of GIST in 1995 and 1998 to 2003 [From the PALGA, the nationwide network and registry of histo- and cytopathology in the Netherlands , all excA clinical centre active in GIST was defined as a centre with more than 15 new pathology diagnosis of GIST per year and a dedicated multidisciplinary sarcoma team. Five Dutch centres met these criteria: the Erasmus Medical Centre, Rotterdam, the Antoni van Leeuwenhoek Hospital, Amsterdam, the University Medical Centre Groningen, the Radboud University Medical Centre, Nijmegen, and the Leiden University Medical Center.and mitotic rate were categorised into to the categories used in the various risk classifications, i.e. < 2, 2–5, 5–10, > 10 cm and 0–5, 6–10, > 10 mitoses per 50 HPF or 5 mm2, depending on what was reported.Data was collected on age at diagnosis, sex, year of diagnosis, localisation, tumour size, mitotic rate, immunohistochemical staining results , mutation analysis and surgical resection margins. Tumour size For the analysis of the different risk stratification scores, patients were grouped according to the criteria of Fletcher et al. , MiettinΧ2 test for trend. Spearman’s rank correlation coefficient was used to test the correlation between the different risk classifications.The incidence rate of GIST was calculated per million inhabitants, also standardised for 5 year age groups and sex for the Dutch population of 2012 and standardised to the WHO and European (ESR) standard population , 37. TimFigure The mean age of patients was 65 years SD 13), median 67 years (range 3–96) and 1307 (53.2%) patients were male , colon (n = 1) and intra-abdominal not further specified (n = 1).Of the six patients < 21 years of age, four were female  and two were male (14 and 17 years). Localisations were the stomach (p < 0.05). Age of peak incidence was 70–74 years with an incidence of 73.9 per million in 2012 for this age group. The incidence of GIST before the age of 21 was 0.13 per million per year (Table The standardised incidence rate increased from 12.2 per million in 2003 to 17.7 in 2012 (p < 0.0001) from 4.0 to 13.5% with at the same time a decrease in the proportion of patients for which tumour size is not reported from 47.1 to 34.8% (ns) with a stable absolute number  with known morphology, 81.6% had spindle cell morphology, 9.3% epithelioid subtype and 9.1% mixed epithelioid/spindle cell subtype. No differences in morphology were found for the specified localisations. For GIST patients < 21 years, histologic subtype was mixed morphology in two, epithelioid subtype in two and unknown in two patients.Immunohistochemistry results were analysed for patients with full pathology reports. CD117 was reported in 89.4% of patients, and of these, 93.6% tested positive. For DOG1, 42.9% of patients were tested with a positive result in 98.6%. For additional results of immunohistochemistry, see Supplementary Table p < 0.001) with each other, with an R ranging from 0.808 (Gold vs Joensuu) to 0.957 (Miettinen 2002 vs Miettinen/AFIP) . Although comparison of the incidence of risk categories is difficult because risk classifications differ in the number of patients eligible for risk stratification, both the Gold risk assessment and the Miettinen 2002 classification seem to allocate more patients to the highest risk group compared with the other risk classifications. All risk classifications had a significant and good to very good correlation (P) Table .Table 2Dp = 0.000).Mutational status was reported in 461 of the 2456 patients (18.8%) based on excerpts and in 166 of 489 patients (33.9%) based on patients with full pathology reports. The presence of PDGFRA mutations is relatively high with a frequency of 16.3%. Supplementary Fig. p = 0.000)  to the high risk group (67.1%)  of GIST in 2011 and 2012 . If this cut-off of > 15 pathology specimens of GIST/year is used as definition of a GIST reference centre and with inclusion of all the regional soft tissue pathology panels, then for 13.2% of patients, the primary diagnosis was established in a GIST centre, surgery was done in 16.2% of the patients in a GIST centre and 35.9% of the patients were diagnosed or had a revision of their diagnosis within 3 months in a GIST reference centre. No significant increase was found in the number of pathology revisions over the years of study , although there seems to be an increasing trend in the number of reviews after the guidelines of 2010   and lastWe can only hypothesise about the cause of the increase in The Netherlands. First, it could be an increased use of diagnostic procedures such as CT scans, gastroscopy and endoscopic ultrasound, which is supported by the increase in number of patients with a small tumour size. Another possible reason is an increased awareness of the diagnosis after the introduction of imatinib as effective treatment. The last possibility could be a real increase in the incidence; although this is a possibility, until now no causal factors or risk factors for the development of GIST are known.The difference in crude incidence for 2003 in the Goettsch paper  and our The incidence of 17.7 per million inhabitants is to the upper limit of reported incidences, although comparison is hampered by a lack of standardised incidence rates –10. ThisThe ESMO guideline of 2010 recommends to perform mutation analysis in all GISTs, because mutational status is related both to prognosis and efficacy of treatment. However, only a minority of patients in 2011 and 2012 (33.9%) had mutational status reported. , 17, 27 In the past, risk classification was not incorporated in the guidelines, and so, mitotic rate and size were often not reported in the conclusion. To get a better overview of the risk classifications, we requested full pathology reports for all patients with a resection in 2011 and 2012. Comparing the different risk classifications, it seems that the Gold and Miettinen 2002 criteria allocate more patients to the highest risk category compared with the other known risk stratifications, but comparison is difficult because these classifications do not include exactly the same patients in our analysis. For example, both the Joensuu and the Miettinen 2002 criteria do only provide stratification rules for gastric and intestinal tumours. Also, the number of risk groups differs between classifications. These factors hamper comparison of the different stratifications.Since 2008 the ESMO guideline recommends mutation analysis for all GISTs and the 2010 guidelines recommends revision of pathology by an expert pathologist, we here show that in 2012 only 41.2% of patients had a revision of pathology within 3 months and only 29.4% of patients had mutational analysis performed. This was much better for high risk patients (based on the Miettinen/AFIP classification) with 67.1% for both mutational analysis and pathology review.In conclusion, this is the second nationwide GIST incidence study ever performed in the Netherlands and follows the previous study in the Netherlands in 2003 . It showESM 1(DOCX 54.4 kb)ESM 2(DOCX 263 kb)ESM 3(DOCX 13.5 kb)ESM 4(DOCX 12.7 kb)ESM 5(DOCX 13.4 kb)ESM 6(DOCX 12.2 kb)ESM 7(DOCX 12 kb)


Phosphorus management through dietetic therapy is vital for the prevention of cardiovascular disease in chronic kidney disease patients. There are two main sources of phosphorus in the diet, organic phosphorus from protein and inorganic phosphorus from food additives. The adverse effects of high phosphorus intake on vascular-endothelium function have been reported; however, the differences in the effects of organic phosphorus versus inorganic phosphorus are not clear. In this study, we examined an acute effect of these high phosphorus meals intake on vascular-endothelium function. This was a randomized, double-blind, cross-over test study design targeting healthy young men. We conducted a food intake test using two test meals, one high in organic phosphorus from organic food sources, and one high in inorganic phosphorus from food additives. Endothelium-dependent vasodilation, phosphorus and calcium in the urine and blood, and phosphorus-related hormones were measured preprandial to 120 min postprandial. The results showed higher serum and urine phosphorus values after the high inorganic phosphorus meal, and a significant reduction in endothelium-dependent vasodilation at 30 min postprandial. These findings are evidence that inorganic phosphorus has a stronger influence on vascular-endothelium function than organic phosphorus. Rather, there are problems with excessive P uptake since it is used abundantly as an additive in processed foods. When renal function is normal, excessive P is excreted in the urine. However, since the renal function of dialysis patients is inadequate, excretion of P is insufficient, and hyperphosphatemia can easily develop.,10P intake from meals is classified into organic P from natural foods and inorganic P from food additives such as pH control agents, coagulants and stabilizers. In healthy people, the rate of absorption of organic P is about 40–60% and that of inorganic P is 90% or more.–4 Therefore, P management through dietetic therapy is important for dialysis patients. However, the high amounts of inorganic P found in food additives make P management difficult. In addition, there are no standardized criteria for inorganic P on food packaging labels in Japan and food manufacturers are under no obligation to provide this information, making it very difficult to ascertain the amount of P intake.Hyperphosphatemia has been associated with arteriosclerosis and vascular calcification, and is an important factor in the onset of CVD in dialysis patients. Vascular endothelial cells exist in the innermost layer of a vessel, and emit various substances essential to vessel operation, such as nitric oxide (NO), endothelin, C-type natriuretic peptide, and prostaglandin I2. Moreover, NO controls the cohesion and flocculation of thromboplastids, functions that protect vessels by preventing blood coagulation. It has been reported that a high P state causes an incremental increase in oxidative stress, a reduction in NO yield in systemic endotheliocytes, and induces an apoptotic state in CKD patients.–16 When damage to endotheliocytes continues over the long term, the protective effect of a vessel fails and CVD due to arteriosclerosis develops.The dysfunction of vascular endothelial cells is an important effect of hyperphosphatemia. The results showed serum P levels were elevated after a high P intake, and serum P level and vascular-endothelium function had a negative relationship. These results are evidence that P from oral intake increases serum P concentration and affects vascular-endothelium function.A previous report investigated the influence of transient P intake by studying the effects of low and high P loading  on vascular-endothelium function in healthy men using a P loading test for inorganic P.In this study, we hypothesized that inorganic P has a stronger influence on vascular-endothelium function than organic P and is connected with CVD. We investigated the differences in phosphate metabolism and the effects of consuming meals high in organic P and inorganic P on vascular-endothelium function.We conducted a food intake test for organic and inorganic P in ten healthy young men without hypertony, smoking history, present illness, or prescription history of vascular-endothelium function-related drugs or antioxidation supplements. The study protocols were approved by the Ethics Committee of the University of Hyogo. Signed written informed consent was obtained from all study subjects before participation. We finally chose six subjects, except men who exceeded standard level of preprandial serum P levels, FGF23 or endothelium-dependent vasodilation. The preprandial characteristics of the six subjects are shown in Table Measurements of stature, weight, percent body fat, lean body mass, and body mass index (BMI) were taken using a digital stature meter AD-6400  or InBody720 Body Composition Analyzer . Measurements of blood pressure and pulse were performed using a fully automatic sphygmomanometer TM-265  before the first food intake test.The study used a randomized, double-blind, crossover design on two different days separated by at least 7 days. The protocol is illustrated diagrammatically in Fig. The subjects came to the laboratory at 08:30 after fasting from 24:00 the previous night and rested in a seated position for about 30 min in a room maintained at 25°C. The two test meals were served in random order. The food intake test was completed within 15 min, and subjects could drink freely thereafter. Measurements were performed for each test meal preprandial (0 min) and postprandial . Measurements for body composition elements, blood pressure, and pulse were taken at 0 min only for the first meal. Vascular-endothelium function was measured as described below, and urine and blood were collected at all four time points. The upper limit  according to the Japanese Dietary Reference Intake 2015 edition was taken into consideration as an amount which does not cause harm to health, and the appropriate P load was determined to be 1,200 mg.The composition of the test meals is shown in Table 2D, and intact PTH were measured at all four time points. Ionized Ca, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides (TG), uric acid (UA), sodium (Na), chloride (Cl), and potassium (K) were measured at three points . All blood test measurements were performed by Hyogo Clinical Laboratory Corporation . Blood glucose level was measured at all four time points using the Glutest Neo Super blood glucose meter  at the time a blood sample was collected. Serum FGF23 concentration was measured using FGF-23 ELISA Kit .Blood samples were collected from the median cubital vein at four time points: preprandial (0 min), and 30, 60 and 120 min postprandial. P, Ca, 1,25(OH)Urine was also collected at all four time points: preprandial (0 min) and 30, 60 and 120 min postprandial. Urine P, Ca and creatinine were measured at all four points. Urine pH, protein, sugar, urobilinogen, occult blood, bilirubin, and ketone bodies were measured at 0 min. All urine test measurements were performed by Hyogo Clinical Laboratory Corporation. Creatinine corrections were calculated for urine P and Ca concentrations in order to eliminate the influence of water intake or sweating.,19 %FMD was measured at four time points: preprandial (0 min) and 30, 60 and 120 min postprandial. If vascular-endothelium function damage occurs, the NO yield and %FMD value will decrease. Generally, the lumen diameter method is used to determine the diameter of a vessel. However, since the subjects of this study were young, the tunica-intima tunica-media composite thickness was thin, and therefore difficult to determine. Instead, we estimated vessel diameter using the diameter between tunica externas for which clear imaging was obtained. The normal diameter measurement between lining membranes is 6% or more; however, using the diameter between tunica externas, the denominator and resting vessel diameter are large, and the %FMD value is determined to be low at about 1%.We evaluated endothelial function by measuring flow mediated dilation , according to previously published guidelines.The %FMD value at each of the three postprandial times was indicated by lumen the rate of change from the preprandial value at 0 min since the preprandial %FMD value varies between individuals.t test or Mann-Whitney U test for variables with a normal distribution. Results for items with a normal distribution are shown as mean ± SD, and/or median (minimum-maximum). We used IBM SPSS Statistics ver. 21.0 for all data analyses and statistical significance was set at less than 5%.The distribution of basic characteristic values, hematometry values, urine analysis values, %FMD values, and the %FMD rate of change was tested using the Shapiro-Wilk test. Verification of the differences between the two meals were determined following quantitative datum using the n = 6) before consuming the first test meal are shown in Table The basic characteristics of the subjects . On the other hand, a significant postprandial change was not seen after the high organic P meal. When comparing the two test meals, the high inorganic P meal resulted in higher serum P at all time points than the high organic P meal. There were no significant changes in serum Ca and ionized Ca (data not shown) at any of the postprandial time points with either of the two test meals, and there were no significant differences between the two test meals.As for HDL-C, LDL-C, the LDL/HDL ratio, TG, UA, Na, Cl, K and blood glucose level, no significant difference was seen between the two test meals at any of the time points. Changes in serum P, Ca and ionized Ca over time are shown in Fig. Changes in urine P and Ca over time are shown in Fig. 2D, intact PTH and FGF23 levels did not show any significant differences between the two test meals at any of the time points.Changes in phosphate metabolism regulation hormones in the blood over time are shown in Fig. p<0.05). However, %FMD values after the high inorganic P meal showed significant reductions at 30 and 60 min postprandial . A tendency for %FMD to remain reduced was observed after the high inorganic P meal compared with the high organic P meal.Changes in %FMD values over time are shown in Fig. p = 0.024).Furthermore, since individual differences were seen at preprandial, postprandial %FMD values are shown as the %FMD rate of change Fig. B. The raWe investigated the influence of two different forms of P on vascular-endothelium function and found that high inorganic P meals resulted in elevated serum and urine P levels, and reduced %FMD values compared with high organic P meals. Therefore, it became clear that inorganic P has a stronger influence on vascular-endothelium function than organic P in healthy young men.,23 As for dietary factors, a high sugar foods, and high salinity foods, high fat diet have been reported to reduce vascular-endothelium function.–26 These dietary factors increase oxidative stress and lead to vascular endothelial cell damage, through the activity reduction of endothelial NO synthase (eNOS) by a reactive oxygen species.A reduction in vascular-endothelium function is one of the initial complications of arterial sclerosis. Factors such as diet, age, level of physical activity, and smoking have been reported to influence vascular-endothelium function.,28 Polyphenolics have antioxidant properties and are known to control the reduction of vascular-endothelium function by reducing oxidative stress. Thus, vascular-endothelium function is subject to the influence of temporary sensitivity to dietary factors. The accumulation of these effects is considered to be related to the development or prevention of arterial sclerosis.On the other hand, it has been reported that vascular-endothelium function is improved by the uptake of polyphenolics, such as a cocoa flavonol and curcumin. Furthermore, it has been shown that serum P levels and %FMD values have a negative correlation. In accordance with the elevation of serum P, oxidative stress increases, NO yield falls, and inorganic P stress is caused by damage to the vascular-endothelium function being carried out. Furthermore, it was assumed that inorganic P reduced %FMD values even though the amount of P loading was equivalent because the rate of absorption of inorganic P is higher than that of organic P.One study of dietary P and vascular-endothelium function in healthy men showed elevated serum P levels and reduced %FMD values after the intake of meals high in inorganic P.One of the strengths of this study is that it shows the influence of two different forms of P on the reduction of vascular-endothelium function for the first time. It is thought that the regulatory mechanism through urinary excretion functioned because an elevation in urine P level was shown with an elevation in serum P level. Moreover, the alteration of phosphate metabolism in this study was temporary, so it did not influence P regulation hormones.One limitation of this study is that there were few subjects. Although there were many subjects at the start of the study, those with serum P, FGF23 and %FMD values over the standard level were excluded. The subjects were also healthy young men, so the fact that many of them showed values exceeding the standards may reflect excessive P uptake in the current Japanese society.In dialysis patients, hyperphosphatemia can induce vascular-endothelium damage. It is suggested that the long-term effects of this damage influence to arterial sclerosis, vascular calcification, and the development of CVD. In this study, the elevation of serum P after high inorganic P meal was normal range, and the reduction of %FMD had for the most part recovered by 120 min postprandial. However, it is thought that a habitual overabundant intake of inorganic P may cause dysfunction of the vascular endothelium in healthy persons by repeating the transient damage to the endothelial cells.,30 It has been also reported that %FMD is decreased by indoxyl sulfate, a protein-bound uremic toxin, and leptin, which is elevated in CKD patients.,32 Thus, P is one of the factors relevant to CVD, and this study showed that P, notably inorganic P, management in the diet is important. However, there are reports that only 17.8% of non-dialysis CKD patients in Japan did not know that inorganic phosphorus was contained in processed foods or drinks. In addition, 68.9% of patients did not know that excessive intake of phosphorus would harm. 37% of CKD patients who begin dialysis already have cardiovascular events. Thus, education about phosphorus intake is necessary form the early stage of CKD patients. The amounts of phosphorus in nutrition facts label of foods, in particular processed foods, is considered to be needed for patients to select foods.It has been reported that a reduction in renal function and a reduction in %FMD values are related, and dialysis patients have low %FMD values compared with healthy persons.In this study, it became evident that inorganic P contained in food additives has a strong effect on vascular-endothelium function and contributes to the development of CVD. Therefore, it is recommended that excessive intake of processed foods with high amounts of inorganic P be avoided. Further study of the factors that prevent the impact of inorganic P will lead to improvements in dietary management for dialysis patients and prevention of CVD.


The statistical significances were calculated through an ANOVA test with a (1 - β) power of 0.861. ClinicalTrials.gov: NCT00989248 (22).p>0.01), oxygen consumption (VO2), (p>0.01) and carbon dioxide production (VCO2) (p<0.01). Also, for the same group, in relation to the environment, water versus on land for HB, VO2, VCO2 and oxygen for each heart beat (VO2/HB) all of than (p<0.01). The stages for data collected featured the subject’s performance throughout the experiment, and within the given context, variables rating of perceived exertion (RPE), HB, VO2, VCO2 and VO2/HB (p<0.01) showed significant interactions between test stages and environment. Additionally, there was a significant interaction between the etiology and the test stages for the variables HB, VO2 and VCO2 (p<0.01). Electrocardiographic changes compatible with myocardial ischemia or arrhythmia were not observed. The subjects exhibited lower scores on Borg’s perceived exertion scale in the water than at every one of the test stages on land (p<0.01).Significant differences were uncovered in coronary artery disease group regarding the variables heart beats (HB), (versus on land allow us to conclude that coronary artery disease patients are able to do physical exercise in water and that the physiological effects of immersion do not present any risk for such patients, as exercise was well tolerated by all subjects.This study show that a cardiopulmonary exercise test can be safely conducted in subjects in immersion and that the procedures, resources and equipment used yielded replicable and reliable data. Significant differences observed in water  Physical activity in a warm water pool is traditionally used in rehabilitation facilities with patients who have different pathologies. As exercise in water can be reasonably easy to perform, it may enhance mobility, strength and physical conditioning During immersion at the level of the manubrium, there is compression on surface veins caused by hydrostatic pressure. Blood flow in lower limbs is reversed from downward to upward through the unidirectional valves, first to the thighs and then upward to the abdomen and finally to the chest and to the heart. During immersion up to the iliac crest, changes in volume are not significant, but central venous pressure begins to increase during immersion at the level of the manubrium and eventually gets higher during total immersion Lung blood flow also increases as a result of increased central volume and blood pressure. Average lung artery pressure ranges between 5 mmHg on land and 22 mmHg during immersion at the neck level. Most of the lung blood volume is directed to the larger vessels of the lung vascular bed Several studies have assessed and validated the use of aquatic therapy for cardiovascular rehabilitation following heart failure and ischemic heart disease. Common sense clinical opinion is that immersion in warm water and controlled exercises are well tolerated by patients diagnosed with coronary artery disease (CAD), even when they experience the physiological effects discussed herein. The assumption is that there are no major differences between a CPX conducted in water and its counterpart on land. Additionally, the required equipment and resources are supposed to be similar, provided that there is compliance with the required test preparation methodology for the CPX conducted in water.versus on an underwater treadmill, to assess the cardiorespiratory performance of coronary artery disease (CAD) patients while immersed in warm water and to compare with the performance of healthy (H) individuals.Thus, the aim of this study was to compare responses to a cardiopulmonary exercise test (CPX) on land The sampling population of this study comprised 40 subjects, 20 of whom were healthy controls and 20 of whom were patients enrolled in the physical conditioning program of the Institute of Medicine and Rehabilitation, Clinics Hospital, School of Medicine, University of São Paulo. The patient group had a medical history of one or more of the following conditions: acute coronary syndrome, coronary angioplasty, revascularization surgery or CAD. Patients were between 55 and 80 years old, and they were first examined by a heart specialist who assessed their clinical history and validated their medical conditions. Any patient who did not comply with the test schedule or those who had peripheral artery disease, diabetes, chronic lung disease, hypertension (blood pressure >160/90), or significant or unstable cardiovascular morbidity conditionswhile taking medication were excluded from the study . ClinicaBoth groups underwent two CPX tests and were always monitored by the same cardiology specialist throughout the testing and that did the data interpretation. All of the participants received clear explanations about the test methodology and granted their consent, after which they were requested to sign an informed consent.3 of water volume and depths ranging between 1.10 and 2.10 m. The nursing staff prepared the subjects for both tests by first cleaning the skin areas where electrodes were applied, connecting the cables, putting the facemasks on the subjects and finally checking their systolic and diastolic blood pressure (SDBP). The gas analyzer was calibrate prior to every run of the test.Clinical data were then collected, and body mass and height were measured. Within an interval of 3-7 days after the land CPX was conducted, the second CPX was conducted in a warm indoor pool with 48 mOn land, the environment temperature was kept stable at 20±2°C and the relative air humidity was maintained at 50-70% Due to a lack of interfaces connected directly the under-water treadmill to a computer, the Bruce Protocol 2), carbon dioxide production (VCO2), oxygen equivalents (VE/VO2) and carbonic dioxide equivalents (VE/VCO2) as well as the respiratory exchange rate (RER - VCO2/VO2).A set comprising a facemask covering the nose and mouth, a cap and a flow sensor was used to conduct the air exhaled by subjects to the gas analyzer. An model Metalyzer II (Cortex CPET) measured respiratory and metabolic variables. As this device was connected to the Ergo-Elite software on the computer, it supplied the following data: exhaled gas (average min), oxygen consumption  reached ≥1.1. After the test was completed, subjects kept walking on the treadmill for 2 minutes at a speed of 2.7 km per hour and 0% incline while still being monitored. The test was interrupted whenever any of the criteria set by the Brazilian Cardiology Society/American Heart Association were not complied with 2 rates increased and metabolism changed from aerobic to anaerobic. This variable shows individual capacity for exercise and the physiologic responses. Three, respiratory compensation point (RCP) showed the aerobic and anaerobic rates. This is the point when the individual is no longer able to endure lengthy exercise as his or her capacity to remove lactate decreases and the anaerobic system takes over the aerobic system. Four, maximum effort (ME), a marker of cardiopulmonary system limits. Five, recovery (REC) showed heart rate recovery rates, any cardiac stress and the rate of metabolic acidosis after exertion that leads to faster and deeper breathing, as the body tries to eliminate excessive acid by reducing the amount of carbon dioxide , accelerated ventilation, heart beat and oxygen consumption were observed to indicate whether subjects were anxious or experiencing metabolic disorders. Two, the anaerobic threshold (AT) featured an exertion level at which CO dioxide .For safety purposes, the experimental environment featured an emergency exit, a team of experts, continuous supervision and monitoring, and an emergency cart equipped with a defibrillator and emergency drugs.Experiment profile for the five stages, environment and study group in each variable .2, VCO2, VO2/HB, VE/VO2, VE/VCO2, and VCO2/VO2. The environment intra-group dependent variable stands for both the on land and in water experiment, and the etiology inter-group independent variables are CAD patients and H individuals. As this was not a treatment in itself, the analogy with a control case experiment was chosen.The stage intra-group dependent variables for both on land and in-water tests are RPE, HB, VOA variance analysis of repeated measurements (rANOVA) was conducted for factors and for every one of the dependent variables. Time gaps between the stage variables were considered equivalent and were not compromised by the intra-group error term. As non-homogeneous variances were expected throughout the test stages, Greenhouse-Geisser or Huyn-Feldt corrections As there is no canonical methodology for identifying significant differences in this type of variance analysis, it was decided to use t-tests for every one of the possible combinations between the three factors under investigation and Holm-Bonferroni corrections for multiple tests. In both cases, the significance level was set at 5%.The sampling population profile is shown by tables with descriptive statistics (means and standard deviations) and is illustrated by graphs of the mean profiles and boxplots for every factor. ANOVA statistical power was calculated at (1 - β)=0.861, thus totaling a minimum of 20 participants per group.This study was approve by the ethical review board of school of Medicine – University of Sao Paulo, protocol number 0532/08. All subjects agreed to volunteer for the study and signed an informed consent form.versus Health one for the variables HB, VO2 and VCO2, all of than with (p>0.01), no significant effect for the other variables. The major effect for environment, land versus water, showed significance for variables HB, VO2, VCO2 and VO2/HB, all of than with (p>0.01)  .p>0.01), HB (p=0.02), VO2 (p>0.01), VCO2 (p>0.01), and VO2/HB (p>0.01), suggested significant interactions between stage and environment. In other words, the on-land mean profiles were different from the in water profiles throughout the experiment. Moreover, there was a significant interaction between etiology and stage for variables HB (p>0.01), VO2 (p>0.01), and VCO2 (p>0.01). CAD patients and H individuals showed different performances throughout the experiment for these variables. There were no significant interactions regarding the others variables. Furthermore, there was no significant interaction between etiology and environment; these factors can be interpreted independently, which there for e justifies the method chosen for the variance analysis and data interpretation . ECG changes compatible with myocardial ischemia or arrhythmia were not observed, and the SDBP did not vary significantly.The Bruce Protocol determines changes in the treadmill incline and speed every 3 minutes, thus resulting in changes in the variables under investigation in the 5 specific set-points of data collection. Hemodynamic changes and cardiovascular overload caused by immersion might lead to a shorter exertion period in the in water test. However, the opposite was observed for both CAD patients and H individuals; the activity time in water was longer than on land  for both groups in every stage. Christie et al., 1990 2 reaches 60% of VO2peak. Our findings demonstrated that the heart rate exhibits significant effects and interactions between etiology and stage, (p<0.01), stage and environment (p<0.01). At all of the stages, the HB rate was higher on land (p=0.02) than in water (p<0.01) for both groups and healthy subjects had a higher HB rate than CAD patients in both environments. This can be explained by the use of medication - CAD group.The resistance of the water, however, which is 800 times denser than air, should theoretically require more effort to walk or jog in on an underwater treadmill. Nevertheless, this study shows quite the opposite. As subjects walked or jogged on the underwater treadmill, they did not fight the resistance of the water, and up thrust became a facilitating force as it demanded less exertion of the anti-gravitational muscles to support their body weight. Consequently, the perceived exertion scale Borg RPE showed statistically significant differences between in water and on land tests . VO2 on land was higher than in water at all the stages for both CAD and Healthy subjects and both showed lower VO2 in water than on land (p<0.01). Craig and Dvorak, 1969 2 was similar for healthy subjects on land and in water at 34°C. This finding, however, could not be replicated in the current study, which suggests that the resistance of water or the temperature (34°C) affected oxygen consumption. CO2 production shows a causal relationship with O2 and a linear increase.VO2 pulse VO2/HB indicates the amount of O2 transported during every heart beat. Lower VE performance may be detected in an increasing exertion test by observing the VO2/HB, and it represents an indirect index of O2 transported by the cardiopulmonary system. Significant effects and interactions were observed for stage and environment (p<0.01), but not for etiology and stage (p=0.75). According to Hall et al., 1990 2/HB, which was lower in water than on land for both groups (p<0.01). However, it does not explain why CAD patients showed lower O2 pulse (p<0.01) in water and on land than healthy individuals.According to Yazbek et al., 1998 2 and VE/VCO2 with VE at body temperature pressure saturated (BTPS) conditions and VO2 and VCO2 at standard air temperature and air pressure dry (STPD) conditions show how many liters of air per minute are necessary and must be ventilated to consume 100 ml of O2 and to produce CO2.The relationships of VE/VO2 at each of the stages. In a previous study, Craig and Ware, 1967 2.This study has assessed that even during increasing exertion and under the physiological effects of immersion, both CAD patients and H individuals did not show significant changes in VE, which remained appropriate to the relative demand of O2 and VO2. During increased exertion, which was the case in this study, metabolism increasingly uses carbohydrates as a source of energy, reaching ≥1.1 at maximum exertion. In both tests (in water and on land), subjects reached a QR>1, so there was no significant effect between stage and environment and etiology and environment.Respiratory quotient or QR reflects the quotient between VCOAs hemodynamic responses to exercise vary depending on cardiac output and peripheral resistance, increased systolic blood pressure was expected as a result of higher cardiac output in both environments, mainly during immersion. Diastolic blood pressure, in contrast, was expected to remain stable or even to mildly decrease with exercise. However, no significant SDBP response was observed. Studies concerning the SDBP responses of healthy individuals during rest at thermo neutral temperature immersion are conflicting and scarce in the literature Gabrielsen et al., 1993 No significant arrhythmia or ventricular ectopic activity and/or instances of ST-segment depression were observed by the cardiologist in the ECG recordings performance evaluation curve.The use of an underwater treadmill without any computer interface prevented this study from automatically controlling the treadmill speed and incline. This might have affected data collection for the in water test, however, the methodology used for both tests was identical and the subjects had to walk or jog in compliance with the Bruce Protocol The order of the test was not randomized because a CPX in water is not well known and, for safety, we prefer to start with a CPX on land, secure and widely used procedure.This study can be presents a small study population although we performed more 144 tests, but many had to be discarded because of the difficulties in collecting data, primarily in the immersion test. That is why we used the statistical power was calculated at (1 - β)=0.861, thus totaling a minimum of 20 participants per group.versus on land allow us to conclude that CAD patients are able to do physical exercise in water and that the physiological effects of immersion do not present any risk for such patients, as exercise was well tolerated by all subjects. We can also conclude that CPX conducted in water may be used by cardiologists to safely diagnose and estimate the exertion capability of CAD patients to perform dynamic exercise in a pool. Further investigation is necessary to provide a clearer understanding of the physiological effects of immersion associated with higher risk subjects.This study show that a CPX can be safely conducted in subjects in immersion and that the procedures, resources and equipment used yielded replicable and reliable data. Significant differences observed in water A cross-sectional study where the effects of medium and long term are not known, but this was not the objective of the study.Garcia MK was responsible for the study conception and design, analysis and interpretation of data and manuscript writing. Rizzo L was responsible for the coordination of the research, conduction of the experiments and manuscript writing. Yazbek-Júnior P was responsible for the conduction of the experiments with cardiopulmonary stress exertion analysis and interpretation of data. Yutiyama D, Silva F.J and Matheus D were responsible for the conduction of the experiments. Mastrocolla LE was responsible for the analysis and interpretation of the findings. Massad E was responsible for the study conception, design, analysis and interpretation of the findings.


To evaluate the correlation between central venous pressure (CVP) and inferior vena cava (IVC) diameters measured by ultrasonography (Ultrasound) in critically ill patients.Intubated critically ill patients were enrolled. The CVP values were measured using a U-tube manometer and were compared to the IVC diameters and collapsibility index, which were measured by bedside Ultrasound. Patients younger than 18 years old, who were not intubated, who had an abdominal pressure greater than 12 mmHg, and/or who were admitted for trauma were excluded from the study.Eighty three patients with a mean age of 73.6±11.2 years were enrolled. The most common diagnosis was sepsis . IVC inspiration measurements were statistically significantly correlated with CVP measurements . IVC collapsibility measurements showed a negative correlation with CVP measurements .There is a strong correlation between CVP and IVC diameters and the collapsibility index. This is a new formula for evaluating CVP, based on our statistical analyses. Fluid resuscitation in critically ill patients is a common and serious challenge.3CVP refers to the pressure of the right atrium or superior vena cava and helps inform emergency departments and critical care units about what fluid and diuretic treatment to apply.6Ultrasound is a good method for noninvasive hemodynamic motorization by emergency physicians, and it may be more helpful than other noninvasive methods such as measuring urine output, pulse rate, and arterial blood pressure. New technological improvements have made Ultrasound mobile, easy to use and inexpensive.7Trans-abdominal Ultrasound measurements of the IVC are noninvasive and thus are not associated with complications. In addition, many emergency departments have Ultrasound systems that can easily be used by emergency physicians.This prospective observational study was conducted at a tertiary care hospital emergency department. Our emergency department serves 360,000 patients annually with a 15% admission rate. Approximately 5% of the patients are evaluated in the critical care area. Hospital ethics committee approval was received for this study (B104ISM4340029/3). An informed consent was obtained from patients because they were intubated. A data collection form was used to gather standard data. Patient age, gender, final diagnosis, arterial blood pressure, pulse rate, CVP measurement at the end of expiration, IVC diameters during expiration and inspiration, and intra-abdominal pressure values were recorded. All the measurements were made on intubated patients.Between June 1 and October 25, 2012, patients who were treated in the critical care area were included in the study. All of the patients were mechanically ventilated using the Synchronized Intermittent- Mandatory Ventilation (SIMV) Volume Control mode and had a central venous line. Patients who met the following criteria were excluded: younger than 18 years old, not intubated, having had trauma, being pregnant, and/or having known or newly diagnosed intra-abdominal hypertension is defined as an intra-abdominal pressure greater than or equal to 12 mmHg.The CVP, intra-abdominal pressure, and IVC diameters were measured in all patients. IVC diameters were measured by emergency department residents and specialists who received a standard basic Ultrasound course for two days. Ultrasound was performed using a Toshiba Aplio500 Ultrasound device and 3 mHz convex probe. The M-mode subxiphoidal window used to view the IVC, and both ends of the inspiration and expiration diameters were recorded in millimeters .2O.The CVP measurements were recorded from the fourth costal cartilage intersection point with the midaxillary line taken as a reference point. The CVP was measured using a U-tube manometer. The CVP results were recorded at the end of expiration in cmH2O and converted into mmHg (1mmHg= 1.36 cmH2O).9The bladder was drained using a urinary catheter before measuring intra-abdominal pressure, and 50–100 mL normal saline was injected into the bladder; the distal portion was clamped. A 16 gauge needle was inserted into the output of the urinary catheter. The needle was connected to a three-way tap and water manometer. Symphysis pubis was taken as a reference point. After being filled with saline, the patient side of the manometer was opened. The intra-abdominal pressure results were measured and recorded in cmHThe NCSS Number Cruncher Statistical System 2007 and Power Analysis and Sample Size 2008 Statistical Software  program were used for the statistical analysis of the data. Data analysis included descriptive statistics; to assess the differences between contineous variables, a Student’s t test was used. The normal distribution of variants was evaluated using Shapiro-Wilk and Kolmogorov-Smirnov analyses. Pearson and Spearman correlation analyses were used to assess correlations between CVP and IVC diameters. To assess the effects of measurements of IVC inspiration and expiration on CVP, multivariate regression analysis was used. The results were evaluated with 95% confidence intervals (CIs), and statistical significance was set at a p value less than 0.05. The data are presented as the mean ± standard deviation (SD) for continuous variables with ranges in parentheses.2O, and the maximum value of the CVP measurement was 30 cmH2O. The mean CVP measurement was 10.1±6.7. IVC inspiration measurements showed a statistically significant correlation with CVP measurements , whereas IVC expiration measurements did not . IVC collapsibility measurements showed a negative correlation with CVP measurements   The characteristics of patients are given in  r:0.68) . In the CVP= 8.813+1.489(IVC Ins)+ 1.101 (IVC Exp).2 = 0.519), IVC inspiration and expiration measurements affected CVP at the rate of 52% .The summary model of the regression model is given in We evaluated the correlation between CVP and IVC diameters as measured by Ultrasound in intubated patients, and found a strong correlation between CVP and IVC inspiration diameter and caval index.In general, CVP measurements, pulmonary arterial catheterization, esophageal Doppler Ultrasound, and trans-esophageal echocardiography are the most widely used methods for volume assessments of critically ill patients.6Ultrasound is a noninvasive method with broad applications in cardiac and hemodynamic evaluations. It can be used to assess acute respiratory failure, acute circulatory failure, and cardiac arrest. It can also be used for venous cannulation. Furthermore, IVC diameters can be measured by Ultrasound, and the European Association of Echocardiography recommends quantification as follows: an IVC diameter <2.1 cm that collapses >50% with a sniff suggests a normal RA pressure of 3 mmHg (0–5 mmHg); an IVC diameter >2.1 cm that collapses <50% with a sniff suggests a high RA pressure of 15 mmHg (10–20 mmHg); in scenarios in which the IVC diameter and collapse do not fit this paradigm, an intermediate value of 8 mmHg (5–10 mmHg) may be used.7IVC is the largest vein, with a low pressure in the venous system. Dilation occurs due to changes in veous pressure. Weil17et al.Previous studies have reported that IVC expiration diameter is correlated with volume loss.There were some limitations to our study. The selection of participants was not randomized, which may have affected the results. However, subjects were enrolled without regard to the severity of illness and with no time limitations. In addition, when measuring CVP, we used a U-tube manometer instead of a piezoelectricity transducer. However, it is not easy to obtain a piezoelectricity transducer. It is an outdated method but it has a wide range of use. Finally, another limitation was that many different residents and specialists performed the procedures. Nonetheless, they were instructed about the ultrasound procedure before starting the study, and all measurements were checked by a physician.In intubated patients, there is strong correlation between CVP and IVC diameter during inspiration. We also found a correlation between CVP and IVC diameters and collapsibility. IVC diameter measurement can be used to assess CVP; thus, this could be an effective method for estimating the volume status of patients.


Whether PR prolongation independently predicts new-onset ischemic events of myocardial infarction and stroke was unclear. Underlying pathophysiological mechanisms of PR prolongation leading to adverse cardiovascular events were poorly understood. We investigated the role of PR prolongation in pathophysiologically-related adverse cardiovascular events and underlying mechanisms.We prospectively investigated 597 high-risk cardiovascular outpatients  for new-onset ischemic stroke, myocardial infarction (MI), congestive heart failure (CHF), and cardiovascular death. Vascular phenotype was determined by carotid intima-media thickness (IMT).P = 0.010). After mean study period of 63 ± 11 months, increased PR interval significantly predicted new-onset ischemic stroke (P = 0.006), CHF (P = 0.040), cardiovascular death (P < 0.001), and combined cardiovascular endpoints (P < 0.001) at cut-off >200 ms. Using multivariable Cox regression, PR prolongation >200 ms independently predicted new-onset ischemic stroke , cardiovascular death  and combined cardiovascular endpoints . PR interval predicts new-onset MI at the exploratory cut-off >162 ms .PR prolongation >200 ms was present in 79 patients (13%) at baseline. PR prolongation >200 ms was associated with significantly higher mean carotid IMT  contains supplementary material, which is available to authorized users. First-degree heart block, referred to as prolongation of PR interval beyond >200 ms on the electrocardiogram (ECG), has long been considered a benign functional variation . NeverthIn a prior study of 88 healthy subjects, PR prolongation was found independently correlated with endothelial dysfunction and increased pulse-wave velocity even in the absence of clinically manifest atherosclerotic disease , suggestTherefore, we investigated in this prospective cohort study the role of PR prolongation as an independent predictor in a continuum of pathophysiologically-linked clinical events including ischemic stroke, MI, CHF, and CV death in high-risk CV patients, and the relationships between PR prolongation and indicator of abnormal vascular function.We studied 597 consecutive patients with CAD or risk equivalent recruited from outpatient medical specialty clinics, as prior described [During the study period Dec 2007 to May 2012, occurrence of CV death, new-onset ischemic stroke,MI and CHF were retrieved from the central computerized clinical records archive of all public hospitals. Primary endpoints of the study were carotid IMT and clinical events of CV death, new-onset MI and ischemic stroke. Secondary Endpoints were new-onset CHF and combined CV endpoints, which included occurrences of any primary endpoints or CHF. Baseline demographic, clinical and laboratory assessments, clinical event definitions, and the inclusion/exclusion criteria were prior detailed elsewhere .R = 0.97, P < 0.001).As previously described in details , carotidEach participant had 12-lead ECG assessment after rested in the supine position for 5 min. PR interval prolongation was defined as >200 ms . Widenedp-value ≤0.20  were entered. Findings were compared with aother Fully-Adjusted Model to verify robustness of study findings, such that variables a priori determined to be relevant were included  for incident CV events such that potential confounder variables with P < 0.001) and greater male predominance (P < 0.001). There were no significant differences in history of diabetes mellitus, hyperlipidemia, smoking, physical activity, resting pulse rate, systolic/diastolic blood pressure, fasting glucose, HbA1c, and hsCRP . Lower LDL-cholesterol was noted in patients with PR prolongation (P < 0.001) with no differences in triglycerides and HDL-cholesterol (P > 0.05), which could be partially due to higher prevalence of statin use among patients with PR prolongation, albeit that was not statistically significant (P = 0.41). PR prolongation was associated with higher serum creatinine indicating impaired renal function , and a higher proportion of patients with PR prolongation had prior CAD (P = 0.037) or stroke (P = 0.017), with expectedly more prevalent use of aspirin (P = 0.017) and ACEI/ARB (P = 0.008). No between-groups difference of PR interval were found regarding the use of nodal-blocking agents including beta-blockers ; and CCB . QRS duration was similar between groups (P = 0.14).As shown in Table R = 0.13, P = 0.002). Patients with PR prolongation had significantly higher mean carotid IMT . Other positive predictors for carotid IMT included age, prior stroke and systolic blood pressure. Statin use was independently associated with lower carotid IMT (P = 0.041). Diastolic blood pressure was no longer a significant predictor after adjusting for pulse pressure in relation to systolic blood pressure (P > 0.05). QRS duration was not associated with carotid IMT .In this group of high-risk patients with CAD or risk equivalent, increased PR interval was positively associated with increased carotid IMT (Pearson P = 0.006), CHF (P = 0.039), CV death (P < 0.001), and combined CV endpoint (P < 0.001). PR interval length was not associated with risk of new-onset MI at cut-off >200 ms (P > 0.05) (primary cut-off) but a positive association was observed at the exploratory cut-off >162 ms (P = 0.008). ROC Curve analysis revealed overall significant prediction of PR interval for new-onset MI .After mean study period of 63.4 ± 10.6 months, 21 (4%) patients developed new-onset ischemic stroke; 26 (4%) patients developed new-onset MI; 37 (6%) patients developed new-onset CHF; 27 (5%) patients had CV death and 87 (19%) patients developed any of these adverse CV events as a combined endpoint Table  and b. PP < 0.05). Furthermore, Kaplan-Meier analysis showed that subjects with PR prolongation >200 ms had significantly reduced survival . The Vascular Function Model showed that adjusting for carotid IMT did not materially alter the independent prediction by PR prolongation . Other independent positive predictors included HbA1c and hs-CRP (both P < 0.05). Higher BMI was associated with reduced CV death (P < 0.05). On the other hand, PR interval predicted total mortality with only marginal statistical significance , and that it did not predict non-CV death .Incidence rates for CV death in patients with PR interval ≤ 200 ms or >200 ms were respectively 0.6 and 1.8 per 100 person-years. Univariable analysis revealed that age, resting pulse rate, systolic blood pressure, serum hs-CRP, creatinine, widened QRS > 120 ms, and PR prolongation were all significant positive predictors for CV death . Furthermore, Kaplan-Meier analysis showed that PR prolongation >200 ms was significantly associated with reduced new-onset ischemic stroke-free survival . Adjusting for carotid IMT in the Vascular Function Model, the independent predictive estimate by PR prolongation was only slightly attenuated .Incidence rates for ischemic stroke in patients with PR interval ≤ 200 ms or >200 ms were respectively 0.5 and 1.9 per 100 person-years. Age, serum creatinine, carotid IMT, and PR prolongation >200 ms were all significant predicting variables for new-onset ischemic stroke . However, the primary cut-off of PR interval > 200 ms was not associated with new-onset MI (P = 0.11). Kaplan-Meier analysis showed that patients with PR prolongation >162 ms had significantly reduced new-onset MI-free survival . Other independent positive predictors for MI included age and prior CAD. BMI was associated with reduced new-onset MI (P < 0.05).Incidence rates for MI in patients with PR interval ≤ 162 ms or >162 ms were 0.3 and 1.3 per 100 person-years respectively. Age, prior CAD, resting pulse rate, systolic blood pressure, serum creatinine, history of ACEI/ARB use, carotid IMT, and PR prolongation >162 ms (exploratory cut-off) were all significant positive predictors for new-onset MI . Patients with PR prolongation >200 ms had significantly reduced new-onset CHF-free survival . Age, resting pulse rate, and history of aspirin use remained independent predictors of CHF .Incidence rates for CHF in patients with PR interval ≤ 200 ms or >200 ms were 1.1 and 2.4 per 100 person-years respectively. Age, resting pulse rate, systolic blood pressure, physical inactivity, prior CAD, history of aspirin/statin use, serum creatinine, hs-CRP, carotid IMT, QRS > 120 ms, and PR prolongation >200 ms were significant positive predictors of CHF . The Fully-Adjusted Model yielded similar estimates . Further adjustment for carotid IMT did not materially alter the independent prediction by PR prolongation . Other independent positive predictors included age, prior CAD, hs-CRP and creatinine .Incidence rates for combined CV endpoints in patients with PR interval ≤ 200 ms or >200 ms were 2.6 and 6.3 per 100 person-years respectively. Age, prior CAD, smoking, systolic blood pressure, resting pulse rate, hs-CRP, creatinine, history of ACEI/ARB use, QRS >120 ms, carotid IMT, and PR prolongation >200 ms were all significant positive predictors for increased combined CV endpoints  and negative predictive value (NPV) at specific cut-off points of PR interval in the prediction of each CV event were presented in Additional file This is our first recognized study to show that PR prolongation alone is a strong and independent predictor for CV death, new-onset ischemic stroke and MI, and combined CV endpoints including CHF among patients with CAD or risk equivalent. We also assessed the vascular phenotype of these high-risk CV patients and found that PR prolongation was independently related to increased carotid IMT. Interestingly, despite the stated observations, adjustment for carotid IMT did not materially alter the prediction of PR prolongation for incident CV events, which suggests that atherosclerosis alone does not fully explain the pathophysiological mechanism along the pathway of PR prolongation leading to clinical events.The clinical implications of this study are multi-fold. Firstly, as opposed to the traditional school of thought that considered PR prolongation as a benign ECG feature , this stMechanisms underlying our clinical observation are likely multi-dimensional. Firstly, PR prolongation representing delayed atrioventricular conduction could be an ECG manifestation of CV ageing . AtherosVascular function as indicated by carotid IMT is a strong, independent and consistent predictor of both incident ischemic stroke and CV events overall among healthy subjects from diverse ethnicities . IncreasIt should be noted that from our study, the magnitude of risk associations of PR interval prolongation with new-onset CV events was striking, and that IMT did not fully explain such associations, suggesting that alternative mediating pathophysiological mechanisms could be present. Also, the HR estimates are relatively large. Despite our meticulous efforts in the study design and data analysis to carefully minimize biases or residual confounding, potential risk of bias and confounding cannot be excluded totally. Findings from the exploratory endpoint analyses should also best be verified in another independent study. Furthermore, the study of incident atrial fibrillation was not included in the current study, which should be explored in the future.In this prospective study of patients with CAD or risk-equivalent, PR prolongation strongly and independently predicts CV death, new-onset MI, ischemic stroke, and combined CV endpoints including CHF. Increased risk of MI emerged at a cut-off as low as PR interval > 162 ms. Whether adverse vascular function in PR prolongation is an intermediate phenotype or represent a mediating pathway prior to clinical events warrants further investigations.


We describe AHA utilization pattern according to age and renal function in type 2 diabetes mellitus (T2DM), in real-life conditions.The analysis was performed using the data set of electronic medical records collected between 1 January and 31 December, 2011 in 207 Italian diabetes centers. The study population consisted of 157,595 individuals with T2DM. The AHA treatment regimens was evaluated. Kidney function was assessed by eGFR, estimated using the CKD-EPI formula. Other determinations: HbA1c, blood pressure (BP), low- density lipoprotein (LDL-c), total and high density lipoprotein cholesterol (TC and HDL-c), triglycerides (TG) and serum uric acid (SUA). Quality of care was assessed through Q score.The proportion of subjects taking metformin declined progressively across age quartiles along with eGFR values, but remained high in oldest subjects (i.e. 54.5 %). On the other hand, the proportion of patients on secretagogues or insulin increased with aging . The percentage of patients with low eGFR (i.e. <30 ml/min/1.73m2) taking either metformin or sulphonilureas/repaglinide was particularly high (i.e. 15.3% and 34.3% respectively).In a large real-life cohort of T2DM, metformin or sulphonylureas/repaglinide, although not recommended, are frequently prescribed to elderly subjects with severe kidney disease. Diabetes has been estimated to account for approximately 1.5 million deaths in 2012, with more than 80% of diabetes-related deaths in low- and middle-income countries .Even though a large number of antihyperglycemic agents are approved for type 2 diabetes (T2DM), major T2DM treatment guidelines suggest metformin as first-line drug treatment, and, if glycemic control is not achieved, the addition of a second drug is recommended .2 [Accordingly, in real-life conditions the majority of people with T2DM take metformin alone or in combination, although prescription is not always appropriate with regards to age and renal function limitations –6. In fa2 .Age also needs to be taken into account when choosing antihyperglycemic agents in the clinical setting. This is mainly due to a well known reduction in GFR with aging which may foster the risk of severe hypoglycemic events . To dateIn this context, the large database of the AMD Annals initiative  providesTherefore, aim of this report was to assess antihyperglycemic treatment, mainly focused on metformin use, in a large sample of patients with T2DM, according to age and kidney function in real-life conditions.2. Glycemic control, as well as, lipid parameters and BP levels were fairly good, being mean HbA1c, LDL-c and BP values 7.2± 1.3%, 101±33 mg/dL and 137±18/7±89 mmHg, respectively. Mean eGFR was 76±21 mL/min/1.73m2. In Table Clinical features of the whole study sample are reported in Table As expected, serum creatinine increased and eGFR declined progressively as a function of age. Prevalence of patients with eGFR <60 ml/min/1.73m2 increased across age quartiles form 4.8% in the first to 45.3% in the forth quartile. The proportion of both micro- and macroalbuminuria also significantly increased with age   are highly prevalent among those attending diabetes centers, being in our series more than 50%. Age should be taken into account when choosing the most appropriate AHA, by keeping an eye not only on target HbA1c , 15, whiIt’s worth emphasizing that about half of patients in the highest age quartile (i.e. median age: 80 years) had HbA1c levels below 7.0%. Thus, although a less stringent glycemic control is usually suggested in these patients , our finA little proportion of our patients was on pioglitazone treatment. Given the well know side effects , although not contraindicated, its use decreased in elderly and among patients with low or very low eGFR.As expected, elderly persons had a lower eGFR. In this subgroup of patients, a large proportion (i.e. more than 50%) was treated with sulphonilureas/repaglinide and thus exposed to an increased risk of hypoglycemic events. In fact, a recent survey, investigating in a real-life condition the risk of severe hypoglycemia in 29,485 sulphonilurea treated diabetic patients shown as it was related to older age and decreased eGFR . Older aMainly based on data from the UKPDS sub-study related to overweight patients , nowaday2 and continuation with additional caution and dose reduction whenever GFR declines to 30-45 ml/min/1.73m2.Consequently, to prevent patients from missing beneficial effects of metformin, some health agencies, including the National Institute for Health and Clinical Excellence –3, suggeThis position was also confirmed by Inzucchi et al. in a recent systematic review of 65 studies investigating upon the risk of lactic acidosis associated to metformin use. They concluded for a less strict approach to metformin treatment might be chosen, although with caution, in patients with T2DM and mild or moderate chronic kidney disease entangling lower doses and careful follow-up of kidney function . These A2 were on metformin , and thus at high risk for major complications.Although the use of metformin in our sample declined with age, the frequent inappropriate use of this drug in the elderly clearly stands out from our data. In fact, 13.4% (n=309) of the oldest patients with eGFR below 30 ml/min/1.73m2) was one of the most frequent contraindication [Epidemiological data show suboptimal adherence to different guidelines, which have recommended various kidney function thresholds for metformin restriction in CKD. Our results are in keeping with data from Huang et al. who retrospectively reviewed metformin-treated patients with T2DM admitted to major teaching Hospitals in Australia. They reported that about 31% of these patients received the drug inappropriately, given the presence of contraindications. Kidney failure  but no patient with stage 4 and 5 CKD was taking metformin .The reason why the inappropriate use of metformin is quite diffuse is beyond the aim of this survey. However, the evidence that not all physicians follow clinical practice guidelines  and thatOur study has some limitations as well as several strengths. Among the former, we need to say that the data were collected in the 2011, when the use of new innovative antihyperglycemic agents such as DPP-4 inhibitors, GLP-1 agonist or Glifozin was still trivial. In this regard. it is anyway worth to underline that these new classes of drugs are still underused in Italy . Second,In conclusion, although recent guidelines have taken a less stringent stance about contraindications to metformin treatment, clinical risk associated to the use of this drug remains high and should be avoided in the presence of severe CKD. Herewith reported and discussed data indicating the persistence of a significant degree of inappropriateness in the prescription of this drug, call to action for implementing more suitable use of antihyperglycemic drugs, especially in older patients with kidney dysfunction.In the present report we analyzed a large cohort of patients with T2DM followed-up at 207 diabetes centers in Italy among those affiliated to the Italian Association of Clinical Diabetologists initiative aiming to investigate the use of antihyperglycemic treatment, according to age and kidney function. The centers participated in the study are homogeneously distributed throughout the country. The analysis was performed using the data set of electronic medical records collected between 1 January and 31 December 2011. For the purpose of the analysis, we considered only patients who were ≥18 years old and with data about estimated GFR (eGFR) and albuminuria. The study population consisted of 157,595 individuals with T2DM.Data from all participating centers were collected and centrally analyzed anonymously. The results were internally approved by the AMD Annals scientific committee. The diagnosis of T2DM was made at participating Diabetes Centers according to the American Diabetes Association 2003 criteria. This initiative includes measuring and monitoring HbA1c, blood pressure, low- density lipoprotein, total and high density lipoprotein cholesterol, triglycerides and serum uric acid by high standard auto-analyzers in public laboratories successfully participating in nationwide quality control programs. The use of specific classes of drugs  was also evaluated. Kidney function was assessed by serum creatinine and urinary albumin excretion measurements. GFR was estimated for each patient using a standardized serum creatinine assay and the CKD-EPI formula . To be iQuality of care was assessed through the Q score, which was developed as part of the study QuED and subsequently validated in the study QUASAR .Data are given as mean values ± standard deviation (SD); categorical variables are described as frequencies and percentages. Mixed regression models, with diabetes clinics fitted as random effect to consider possible differences in data across centres, were used to compare patients’ characteristics by groups. Continuous and categorical variables were analyzed, respectively, by linear and logistic mixed regression models. P values of <0.05 were considered statistically significant. The analyses were made using STATA software, Version 14 .


ER-α gene polymorphisms may exert different effects in childhood, we analyzed the associations between the IVS1 −397T>C (PvuII) polymorphism and systemic inflammatory state, proangiogenic factors, frequency of monocyte subsets, lipid profile, blood pressure, and vascular complications in girls with type 1 diabetes mellitus (DM1). We examined 180 young girls with DM1 and 120 healthy age-matched controls. The analysis concerned PvuII polymorphism of the ER-α gene as well as the levels of serum inflammatory markers , proangiogenic factors , 17β-estradiol, values of monocyte subsets (CD14++CD16− and CD14+CD16+), lipid profile, and blood pressure. In our study, girls with CC genotype had lower level of inflammatory and angiogenic factors and lower frequencies of CD14+CD16+ monocytes in comparison to CT or TT carriers. Simultaneously, the CC carriers had a greater population of CD14++CD16− monocytes, increased blood pressure, and serum levels of: estrogen, total cholesterol, triglycerides, and low-density lipoprotein cholesterol than girls bearing CT or TT genotype. Our study suggests a pleiotropic effect of PvuII polymorphic CC variant on diabetic vasculopathies. Although the CC genotype carriers demonstrate less inflammatory and angiogenic activity, they seem to display less favorable cardiometabolic features. Based on our study, we cannot distinguish PvuII ER-α genotype that could be useful in identification of DM1 girls that are more prone to develop of late vascular complications, before the occurrence of first clinical symptoms.The effect of estrogens is mediated by activation of estrogen receptors (ERs). Because  Type 1 diabetes mellitus (DM1) is caused by autoimmune selective destruction of pancreatic β-cells. As a multifactorial disease, it is caused by a complex combination of genetic and environmental factors triggering autoimmunity . IdentifDiabetes-related vascular complications share etiological characteristics. Factors recognized as responsible for the pathogenesis of diabetic vasculopathy are: hyperglycemia, hyperlipidemia, growth factors, hormones, and inflammation . Low graAccumulating experimental and clinical data confirmed the influence of gender on autoimmunity, with women being much more susceptible to autoimmune disorders . MoreoveER-α gene and estrogen-related clinical effects [ER-α gene. Functional mechanisms attributed to the polymorphisms of the ER-α gene include alternation of transcription factors binding sites followed by modified gene expression [Estrogens play an important role in the development and function of male and female reproductive systems. They also exert effects on the immune system , skeleta effects . The mospression  or a chapression .ER-α gene and inflammation as well as vascular complications in DM1 patients. In our previous study [+Foxp3+ Tregs and simultaneous lower TNF serum level in comparison to girls carrying CT or TT genotype. Moreover, elevated levels of analyzed proinflammatory factors  in DM1 girls carrying TT genotype promoted enhanced inflammatory response, which led to development and progression of diabetic microvascular complications [Some studies suggested a link between PvuII polymorphic variants of the us study , we haveications . In anotications .ER-α gene polymorphism on systemic inflammation. Some studies indicated proinflammatory profile in individuals with TT variant of this polymorphism [ER-α gene polymorphism in inflammation. Our previous studies indicated that CC variant of PvuII ER-α gene polymorphism may be connected with attenuated chronic inflammatory response during DM1 [Some genetic investigations were conducted to establish the effect of PvuII morphism , whereasmorphism  or even morphism . Despitering DM1 –21. HoweER-α gene polymorphism is associated with the major contributory factors for vascular complications such as systemic inflammatory state, lipid profile, proangiogenic factors, blood pressure, and vascular complications in girls with DM1.Taking all these into account, we aimed to examine whether the PvuII This case–control study evaluated 180 young regularly menstruating girls with diagnosed type 1 diabetes who were recruited from the Chair and Clinics of Pediatrics, Diabetology and Endocrinology, Medical University of Gdańsk. Mean age of patients was 15.5 ± 3.5 years. The diagnosis of type 1 diabetes was based on the American Diabetes Association criteria . PatientThe control group consisted of age and BMI matched 120 healthy young menstruating girls recruited during control visits in an outpatient clinic. No signs of autoimmune, chronic, inflammatory, or neoplastic disease at the time of sampling and no evidence of DM1 in their families were disclosed as confirmed by medical records, laboratory examination and laboratory tests.The urinary albumin excretion (UAE) was expressed as the average of three 24 h collections. Cases were classified as microalbuminuria when in at least two out of three urine samples, UAE ratio was >30–300 mg/24 h. Diabetic nephropathy was defined as persistent microalbuminuria in two out of three consecutive urine samples without clinical or laboratory evidence of other kidney or urinary tract disease.All of the girls with diabetes-related vascular complications  were newly diagnosed and previously untreated.The blood from all patients was collected in the follicular phase (between days two and four) of menstrual cycle. Additionally, the level of plasma 17β-estradiol was determined in all girls using commercial enzyme-linked immunosorbent assay kits  according to the manufacturer’s protocol.The study was conducted according to the principles of the Declaration of Helsinki. All individuals provided written informed consent to participate in a study. The protocol was approved by the Ethics Committee of the Medical University of Gdańsk.g for 15 min and stored at −70 °C until analysis.Serum samples were collected from all girls and processed by centrifugation at 500×Genomic DNA from all the subjects was isolated from EDTA-stabilized blood using the EXTRACTME DNA BLOOD . DNA was stored at −20 °C until the time of use.Systolic and diastolic blood pressures  were measured using automatic 24 h ambulatory blood pressure monitoring (ABPM) by the Holter method. All the average values of the blood pressure were expressed in the centile charts. Arterial hypertension was diagnosed when the blood pressure value reached at least 95th percentile for the corresponding age, gender, and height on at least three separate occasions .Ophthalmologic investigation was performed in all girls with DM1. Diabetic retinopathy was determined by visual acuity, intraocular pressure measurement, anterior segment estimation by slit lamp , and fluorescein angiography . The eye fundus examination was performed with the +90D lens . Analysis of the eye fundus pictures was based on The International Diabetic Retinopathy Division .Characterization of IVS1 −397T>C of the ER-α genotypes (rs2234693) was analyzed as previously described .DNA samples were first sequenced to establish three ER-α gene polymorphic variants, as a quality control. Next, DNA samples of the CC, CT, and TT individuals were routinely added to the examined ones to ensure genotype accuracy.Plasma cytokines were measured at the time of inclusion in the study. Levels of IL-6, TNF-α, VEGF, and angiogenin were determined using commercial enzyme-linked immunosorbent assay kits  according to the manufacturer’s protocol.++CD16− and CD14+CD16+ monocytes in the peripheral blood of all subjects were evaluated. 50 μL aliquots of fresh venous blood were stained with anti-CD14  and anti-CD16  human antibodies. For each set, appropriate isotype control was done. The samples were incubated for 30 min in the dark at room temperature, lysed, and fixed with Immuno-prep reagents  on Q-prep Immunology Workstation .By means of flow cytometry, the values of CD14Expression of cell surface markers was assessed using flow cytometry  and data were analyzed by FACSDiva 6.0 Software (BD Bioscences). Monocytes were gated according to their forward and side scatter characteristics. Typically, 10,000 events were acquired in this region. The monocytes subsets were identified based on the expression of the CD14 and CD16 molecules from dot plots representing CD14 versus CD16 staining.2 test. Normally distributed variables were analyzed with the one-way ANOVA test. Nominal variables were analyzed by the χ2 Pearson test. For comparison of the nonparametric variables, the Kruskal–Wallis ANOVA test was applied. The level of significance was set at p ≤ 0.05.The results were analyzed using Statistica, ver. 12 . Conformation of the allele frequencies to the Hardy–Weinberg equilibrium proportions was tested by the χp = 0.20). The genotype distributions in the group of healthy girls were: CC—22.5%, CT—52.5%, TT—25% and they also conformed to the Hardy–Weinberg principle (p = 0.58). Comparison of frequencies of PvuII genotypes between the DM1 patients and healthy group revealed lack of significant differences .The frequencies of IVS1 −397T>C genotypes in DM1 group were as follows: CC—23.9%, CT—45%, TT—31.1%. These frequencies conformed to the Hardy–Weinberg equilibrium . Systolic blood pressure was the highest in patients with CC genotype and the lowest in TT carriers. Similarly, CC variants of ER-α gene had significantly higher diastolic blood pressure when compared to CT and TT bearing patients.The clinical characteristics of diabetic patients differing in the IVS1 −397T>C estrogen receptor α polymorphism are presented in Table p < 0.001) when compared to CT and TT carriers.As to estrogen concentration, the level of 17β-estradiol was measured in serum of DM1 girls, with samples being collected between 2nd and 4th days of their menstrual cycle. We have noticed that DM1 girls possessing CC genotype had higher estradiol levels . Moreover, the CC variants of ER-α gene had lower VEGF and angiogenin levels when compared with CT and TT patients .To extend our analysis, we measured the serum levels of inflammation markers  and angiogenic factors  in DM1 girls with different genotypes Table . We foun++CD16− and CD14+CD16+ cells  but not HDL (p = 0.26) when compared to their CT and TT counterparts.The next step of our work was to analyze the association between lipid levels  and the genetic variant of estrogen receptor α gene polymorphism Table . We founER-α gene with respect to hypertension occurrence (p = 0.21).The group of girls with type 1 diabetes was analyzed with regard to hypertension as existing macrovascular complication. The results are shown in Table ER-α genotypes due to diabetic microvascular complications development. Alleles distribution in patients with nephro- and retinopathy was similar in comparison to complications-free group . Among 180 diabetic girls, 31 patients had nephropathy and 22 had retinopathy Tables , 7. HoweER-α gene polymorphism with systemic inflammatory state, lipid profile, proangiogenic factors, blood pressure, and vascular complications in girls with type 1 diabetes mellitus.The increased incidence of autoimmune disorders among female patients and a greater risk of fatal and nonfatal vascular events in DM1 women gave rise to an interest in the regulation of the immune/inflammatory response by sex hormones. In the present study, we analyzed the association of the PvuII ER-α gene polymorphism in a smaller group of girls [ER-α gene polymorphism binds estrogens much more efficiently than TT, which is consistent with studies suggesting that lower estrogen concentration is associated with an increased production of proinflammatory cytokines [Similarly to previous reports regarding PvuII of girls , 20 and of girls  with DM1of girls  have proytokines , 29.ER-α gene polymorphism and blood pressure in DM1 boys, where CC genotype was connected with the lowest values of SBP and DBP [ER-α gene polymorphism may influence blood pressure regulation since estrogens are known to stimulate vasodilation, mainly by inducing production of nitric oxide [In this study, we have noticed that girls with DM1 bearing CC genotype had higher systolic and diastolic blood pressure in comparison to CT and TT genotype bearing counterparts. Interestingly, we have already observed an inverse relationship between PvuII  and DBP . This ob and DBP . PvuII Eic oxide  in an ERic oxide . Moreoveic oxide , 34.Another important observation from the current study is the fact, that girls with DM1 carrying CC genotype had lower VEGF and angiogenin serum level when compared to their CT and TT genotype bearing counterparts. VEGF is a critical component in tissue growth and organ repair processes mediated by angiogenesis and vasculogenesis. As a factor included in blood vessel formation, it also plays a pivotal role in the development of microvascular complications. In some disorders, elevated VEGF level acts as a pathological angiogenic stimulus , whereas in others, low protein activity leads to other undesirable conditions  . Angioge++CD16− and “nonclassical”—CD14+CD16+ cells. We have found that girls bearing the CC genotype had higher frequencies of classical monocytes and lower frequencies of nonclassical cells when compared with CT and TT counterparts. During infection, classical monocytes, comprising ~85% of the whole monocyte population undergo rapid recruitment from the blood stream into surrounding tissues, where they become activated. Nonclassical monocytes are localized within capillaries, small veins, and arteries where they crawl over the endothelium patrolling the vasculature for tissue damage or infection. The CD16+ monocytes undergo expansion in various infectious and inflammatory diseases as well as are involved in pathogenesis of autoimmune disorders [+ cells were termed proinflammatory monocytes [+ monocytes play an important role in promoting inflammatory response in DM1.By means of flow cytometry, we have identified two monocyte subsets in DM1 girls: the “classical”—CD14isorders . In addiisorders . Based oonocytes . Estrogeonocytes . Based oonocytes , 45 it iER-α gene polymorphism with lipid profile are controversial, there are clear proofs that PvuII polymorphism can influence estrogen-dependent lipid variables [Another observation from the current study is the fact, that girls with DM1 bearing CC genotype had higher levels of TC, TG, and LDL but not HDL when compared to their CT and TT counterparts. Various studies have shown a positive correlation between elevated serum lipids  and macrovascular complications . On the ariables .In the present study, the PvuII ER-α genotype distribution within patients with vascular diabetic complications showed lack of statistically significant differences when compared to uncomplicated patients. We can speculate that CC variant of this polymorphism is associated with weaker inflammatory response and angiogenic activity leading to slower progression of diabetic complications. On the other hand, CC variant promotes cardiometabolic risk factors responsible for more rapid progression of vascular dysfunctions in DM1. Taking all of these facts into account, this is possibly the reason why we cannot observe differences in PvuII ER-α genotypes distribution in DM1 girls with and without vascular complications. The list of risk factors examined in our study is not exhaustive and other components may influence the association between PvuII polymorphism and cardiovascular diabetic complications.In conclusion, at this stage of our study, it is hard to clearly determine whether PvuII ER-α genotype polymorphism may be a useful tool for identification of late vascular complications, as its CC variant seems to reduce inflammation which protects against development of complications. On the other hand, the same CC variant seems to impair lipid profile in DM1 patients making them more prone to develop cardiovascular complications.