CLNK rs13144136 at p = 1.00x10-6  in the full dataset with similar results in the European American only dataset. We also examined whether SNPs known to influence blood pressure or hypertension also influenced resistant hypertension. None was significant after correction for multiple testing. These data highlight both the difficulties and the potential utility of EHR-linked genomic data to study clinically-relevant traits such as resistant hypertension.Resistant hypertension is defined as high blood pressure that remains above treatment goals in spite of the concurrent use of three antihypertensive agents from different classes. Despite the important health consequences of resistant hypertension, few studies of resistant hypertension have been conducted. To perform a genome-wide association study for resistant hypertension, we defined and identified cases of resistant hypertension and hypertensives with treated, controlled hypertension among >47,500 adults residing in the US linked to electronic health records (EHRs) and genotyped as part of the electronic MEdical Records & GEnomics (eMERGE) Network. Electronic selection logic using billing codes, laboratory values, text queries, and medication records was used to identify resistant hypertension cases and controls at each site, and a total of 3,006 cases of resistant hypertension and 876 controlled hypertensives were identified among eMERGE Phase I and II sites. After imputation and quality control, a total of 2,530,150 SNPs were tested for an association among 2,830 multi-ethnic cases of resistant hypertension and 876 controlled hypertensives. No test of association was genome-wide significant in the full dataset or in the dataset limited to European American cases  and controls (n = 708). The most significant finding was  Hypertension, or high blood pressure, affects nearly a third of the US adult population and is a major risk factor for coronary heart disease, stroke, and kidney disease , 2. PharAlong with age and obesity, well-established independent risk factors of resistant hypertension, several other lifestyle and biological risk factors are believed to contribute to resistant hypertension, including excess alcohol use, increased dietary sodium intake, and use of several classes of medications  . GeneticTo help overcome the obstacle of limited sample size, we used data available from seven electronic health record (EHR) systems to identify both resistant hypertension cases and hypertensive individuals who responded well to a single antihypertensive medication (controlled hypertensives) in the electronic MEdical Records & GEnomics (eMERGE) Network , 23. We The eMERGE Network and its studies are approved by the Institutional Review Board at each study site, which include Geisinger Health System, Group Health, Marshfield Clinic, Mayo Clinic, Mount Sinai School of Medicine, Northwestern University, University of Washington, Vanderbilt University , 23. ParThe eMERGE Network, initially funded in 2007 by the National Human Genome Research Institute (NHGRI), consisted of five EHR-linked biorepositories and a coordinating center at the initiation of this study . In brieIn the second phase of eMERGE, the number of study sites expanded to include Children’s Hospital of Philadelphia, Boston Children’s Hospital, Cincinnati Children’s Hospital Medical Center, Geisinger Health System (GHS), and Mount Sinai School of Medicine (MSSM) . In thisThere are multiple definitions of resistant hypertension in the literature , includiDefining any resistant hypertension group within the EHR requires a patient’s hypertensive status pre- and post- antihypertensive medication use (based on issued prescriptions) and the medication classes of the anti-hypertensives prescribed. To capture these data, two algorithms were deployed in the eMERGE Network to identify resistant hypertension cases. For the first case algorithm (“controlled case”), each case required the concurrent use of at least four antihypertensive medication classes. Medication classes considered for these definitions included angiotensin converting enzyme inhibitors or angiotensin receptor blockers, beta blockers, non-dihydropyridine calcium channel blockers, dihydropyridine calcium channel blockers, hydralazine, minoxidil, central alpha agonists, direct renin antagonists, aldosterone antagonists, alpha antagonists, and diuretics  . Direct For the second case algorithm (“uncontrolled case”), each case required the concurrent use of at least three antihypertensive medication classes and a systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg for at least one month after meeting the medication criteria. For both case definitions, patients were excluded if they had systolic heart failure (defined as an ejection fraction ≤35%) or chronic kidney disease. Chronic kidney disease was defined as an estimated glomerular filtration rate ≤30 ml/min as calculated using the Modification of Diet in Renal Disease formula [Controlled hypertensives (the “controls” for this association study) were defined as patients with a measurement of systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg prior to meeting the medication criteria or with ICD-9 codes for hypertension (401.*) at any time and have one medication from the medication classes described above , and have all systolic and diastolic measurements <135 mg/dl and <90 mg/dl, respectively, one month after blood pressure medications were prescribed (this requires at least one blood pressure measurement). For both cases and controls, simultaneous medication class usage was defined as evidence that the patient was taking the medication concurrently based on the presence of the medications in the same medication list  or via medication refill data with accompanying evidence of overlapping prescriptions for each drug. Like the resistant hypertension cases, individuals with systolic heart failure or chronic kidney disease were excluded.Electronic selection logic using billing codes, laboratory values, text queries, and medication records was used to identify resistant hypertension cases and controls at each site . The iniGenotyping was performed for eMERGE I study sites using two Illumina arrays at two genotyping centers. Individuals of self-identified or administratively-assigned European-descent were genotyped on the Illumina 660W-Quad, while individuals of self-identified or administratively-assigned African-descent were genotyped on the Illumina 1M. For the majority of patients, genotyping was performed at one of two centers: the Center for Inherited Disease Research (CIDR) at Johns Hopkins University and the Center for Genotyping and Analysis at the Broad Institute as previously described , 34. ExiQuality control (QC) measures were developed by the eMERGE Genomics Workgroup  and wereAll genotype data were imputed by Pennsylvania State University Center for Systems Genomics as part of the eMERGE Coordinating Center as previously described . Briefly2), genotyping platform, and genetic ancestry . Analyses were performed in the combined eMERGE I and eMERGE II datasets . The models did not converge in the combined dataset, and inspection of the distribution of cases and controls by study site and genotyping platform revealed several strata with only case counts but no control counts. Removal of the case-only counts (231) enabled the models to converge.For the GWAS, single-SNP tests of association were performed in PLINK  and PLAThttp://www.gettinggeneticsdone.com/2011/04/annotated-manhattan-plots-and-qq-plots.html), and regional plots were generated using LocusZoom at default settings [The results of the GWAS tests of association were plotted using a Manhattan plot. Manhattan plots were generated by the R statistical package using code provided by the blog “Getting Genetics Done”  [SNPs previously associated with blood pressure, systolic blood pressure, diastolic blood pressure, or hypertension among adults at genome-wide significance were drawn from the NHGRI European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog (er 2016) , 47. We er 2016) .A total of 3,006 resistant hypertension (uncontrolled and controlled) cases and 876 controlled hypertensives were identified from the EHRs in eMERGE I and II . SlightlESR1 rs9479122 at p = 6.28x10-11 with an odds ratio of 0.46  and controls (n = 876) adjusted for sex, decade of birth, median BMI, genotyping platform, and ten PCs. A single association at genome-wide significance was observed between case status and  of 0.46  Figs. WhESR1 rs9479122 in two independent datasets, INVEST [ESR1 rs9479122 is nearly monomorphic , which is in agreement with HapMap CEU estimates. ESR1 rs9479122 is imputed in this eMERGE dataset [To corroborate these potential findings, we performed a look-up for , INVEST , 49 and , INVEST . INVEST  dataset .ESR1 rs9479122 a likely false positive, removed it from the dataset, and re-generated the Manhattan plots . CLNK rs13144136 was not associated with resistant hypertension in either the INVEST or SPS3 datasets (p>0.20). A total of 360 associations had nominal significance at p<10−4 in the present study, of which 14 were associated at p<10−5  with blood pressure, systolic blood pressure, diastolic blood pressure, or hypertension. From published GWAS papers [TBX3 rs35444, previously associated with increased diastolic blood pressure in East Asians [-3) in this multi-ethnic clinical study. With 2,830 cases and 876 controls, we were powered to replicate genetic associations for common variants  with an odds ratio ≥1.5, assuming a genetic additive model and a significance threshold of 0.001. As might be expected given that the SNPs tested were from previously reported GWAS, all but one (SLC39A8 rs13107325) of the 50 SNPs met the minor allele frequency threshold assumed in the power calculations based on the 1000 Genomes Project phase 3 genotype data from 2,500 worldwide individuals [We examined whether SNPs known to influence blood pressure or hypertension also influenced resistant hypertension. The NHGRI-EBI GWAS catalog was used to select SNPs previously associated  [CYP3A5) [GSTM1) [NOS3) [ATP2B1 rs12817819) did not replicate in an independent dataset, but reached chip-wide significance after meta-analysis [This present work expands on earlier studies of the genetics of resistant hypertension. The handful of studies that have specifically investigated resistant hypertension have mostly focused on candidate genes: β and γ subunits of the epithelial sodium channel (ENaC) , cytochr[CYP3A5) , glutath [GSTM1) , and end) [NOS3) , 20. How) [NOS3)  that tes) [NOS3) . The mosanalysis .Major strengths of our study include the scale and longitudinal nature of the EHR. The combination of collaboration among research groups with access to biobanks and advances in high-throughput technology allowed us to genotype hundreds of thousands of genetic variants and impute to more than two million SNPs in >2,800 resistant hypertensive patients, enabling us to test for novel associations that may not have been interrogated via traditional candidate gene studies. However, compared with GWAS of common diseases, studies requiring exposure to medications or other treatments face additional challenges . While GStatistical power has been a major obstacle for studies designed to identify genetic variants associated with blood pressure in general. Indeed, even contemporary studies with access to genome-wide and imputed data of millions of common variants estimate that one-third to one-half the variability observed for systolic and diastolic blood pressure levels can be attributed to the additive effects of many genetic variants; to date, only ~2% of blood pressure variation can be explained by known blood pressure-associated variants , each wiThe upcoming NIH Precision Medicine Initiative Cohort Program (“All of Us”), which seeks to accrue >1 million individuals with longitudinal EHR data, may facilitate future studies on drug effects on a scale not possible today , 81. AlsPhenotypic heterogeneity in general is a major challenge faced by most studies . Our stuOf special note is the issue of poor compliance or non-compliance. Most studies involving medications or self-administered treatments have a problem with non-compliance. For hypertension, clinical and epidemiologic surveys continuously suggest that a high proportion of patients do not achieve target blood pressure levels after treatment , 87. ThiFinally, a major challenge in designing optimal studies to identify and characterize the genetic architecture of resistant hypertension may be related to the as-of-yet unknown etiology of the phenotype(s) . For exaIn conclusion, we describe an approach to identify resistant hypertension cases and controls from seven different institutions’ EHRs. To our knowledge, this is the only GWAS of resistant hypertension in any population. These results highlight the utility of EHR-linked genomic data to further refine our understanding of disease and its subtypes, with the goal of uncovering new therapeutic targets and providing better targeting of current therapies.S1 FigESR1 rs9479122 on chromosome 6, a likely false positive due to poor genotyping for this variant.A total of 876 cases of resistant hypertension and 2,830 controlled hypertensives from the eMERGE I and II Network were available for analysis. Single-SNP tests of association were performed for 2,530,150 SNPs using logistic regression, assuming an additive genetic model, adjusted for sex, decade of birth, median body mass index, genotyping platform, and genetic ancestry . Results of each test of association are plotted as p-values (expressed as–log10 on the y-axis) by chromosome (x-axis). The most significant finding was (DOCX)Click here for additional data file.S2 FigESR1 rs9479122) is likely a false-positive due to poor genotyping prior to imputation and was removed.The most significant finding ((DOCX)Click here for additional data file.S3 Fig10 of the p-value. The most significant finding was ESR1 rs9479122 on chromosome 6, a likely false positive due to poor genotyping for this variant.A total of 2,530,150 SNPs were tested for an association with resistant hypertension  among Europeans from the eMERGE I and II Network. Tests of association were performed using logistic regression assuming an additive genetic model and adjusting for sex, decade of birth, genotyping platform, median body mass index, and principal components (1–3). Each test of association was plotted where the x-axis is the chromosomal location and the y-axis is–log(DOCX)Click here for additional data file.S4 FigESR1 rs9479122) is likely a false-positive due to poor genotyping prior to imputation and was removed.A total of 2,530,150 SNPs were tested for an association with resistant hypertension  among Europeans from the eMERGE I and II Network. Tests of association were performed using logistic regression assuming an additive genetic model and adjusting for sex, decade of birth, genotyping platform, median body mass index, and principal components (1–3). The most significant finding ((DOCX)Click here for additional data file.S5 Fig10 of the tests of association and the right x-axis is the recombination rate (cM/Mb). The most significant result in European Americans (rs9479122) is plotted as the index variant color-coded as a purple diamond. Surrounding SNPs are circles color-coded by strength of linkage disequilibrium , where red is complete or very strong LD and blue is weak LD or independent variants. LD was calculated using HapMap CEU data (release 22) in the default version of LocusZoom. Gene names and position on chromosome 6 (Mb) are given on the y-axis. The most significant finding (ESR1 rs9479122) is likely a false-positive due to poor genotyping prior to imputation and was removed.A genome-wide association study was performed for 1,719 European American cases of resistant hypertension and 708 controls from the eMERGE Network adjusted for sex, decade of birth, median body mass index, genotyping platform, and genetic ancestry . The left x-axis is the–log(DOCX)Click here for additional data file.S6 FigESR1 rs9479122, tests of association were performed using logistic regression assuming an additive genetic model and adjusting for sex, decade of birth, genotyping platform, median body mass index, and principal components (1–3). Each test of association was plotted on a Manhattan plot where the x-axis is the chromosomal location and the y-axis is–log10 of the p-value.A total of 2,530,150 SNPs were tested for an association with resistant hypertension  among Europeans from the eMERGE I and II network. After removal of (DOCX)Click here for additional data file.S7 FigESR1 rs9479122, tests of association were performed using logistic regression assuming an additive genetic model and adjusting for sex, decade of birth, genotyping platform, median body mass index, and principal components (1–3).A total of 2,530,150 SNPs were tested for an association with resistant hypertension  among Europeans from the eMERGE I and II network. After removal of (DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableIndividuals were excluded from case or control status based on ICD-9-CM codes and situations as described. In addition to exclusions based on codes, individuals were excluded from case status if there was evidence of chronic kidney disease within six months after meeting the definition for “controlled” resistant hypertension (four medication classes concurrently) or heart failure within one year before or after meeting the definition for “controlled” resistant hypertension. Chronic kidney disease was defined by an estimated glomerular filtration rate (eGFR) ≤30 ml/min, as calculated by the Modification of Diet in Renal Disease formula. Heart failure was defined as an ejection fraction (EF) or left ventricular ejection fraction (LVEF) ≤35%. Individuals with evidence of heart failure were also excluded from control status.(DOCX)Click here for additional data file.S3 TableCounts within parentheses represent number of additional samples identified as cases or controls but not included in the final analyses due to model convergence issues.(DOCX)Click here for additional data file.S4 Tablehttp://www.ebi.ac.uk/gwas/; accessed September 2016). Published GWAS data are compared with eMERGE I and II association results for resistant hypertension among all racial/ethnic adults if the SNP is present in the dataset. Abbreviations: beta (β), p , odds ratio (OR), lower 95% confidence interval (L95), and upper 95% confidence interval (U95). Data from eMERGE I and eMERGE II are denoted by the subscripts.The “Published GWAS” columns represent SNPs previously associated with blood pressure, systolic blood pressure, diastolic blood pressure, or hypertension among adults at genome-wide significance drawn from the NHGRI European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog ((DOCX)Click here for additional data file.


To assess: (1) the frequency of an abnormally large fall in blood pressure (BP) upon standing from supine in patients with initial orthostatic hypotension (IOH); (2) the underlying hemodynamic mechanisms of this fall in BP upon standing from supine and from squatting.In a retrospective study of 371 patients (≤30 years) visiting the syncope unit, the hemodynamic response to standing and squatting were studied in 26 patients who were diagnosed clinically with IOH, based on history taking only. In six patients changes in cardiac output (CO) and systemic vascular resistance (SVR) were determined, and the underlying hemodynamics were analyzed.15/26 (58 %) patients with IOH had an abnormally large initial fall in systolic BP (≥40 mmHg). There was a large scatter in CO and SVR response after arising from supine . The hemodynamic response after squatting showed a more consistent pattern, with a fall in SVR in all six patients .The clinical diagnosis of IOH is based on history taking, as an abnormally large fall in systolic BP can only be documented in 58 %. For IOH upon standing after supine rest, the hemodynamic mechanism can be either a large fall in CO or in SVR. For IOH upon arising from squatting a large fall in SVR is a consistent finding. Occasional light-headedness and seeing black spots upon standing from supine or arising from a squatting position are experienced in almost all, otherwise healthy, teenagers and young adults –4. In thHowever, it has not been studied how often a large initial fall in BP can be documented in subjects referred with severe complaints of light-headedness and occasionally (near) syncope during formal cardiovascular reflex testing, i.e., the test characteristics of the active standing test for the diagnosis of initial orthostatic hypotension (IOH) are unknown .In the laboratory both active standing from supine and arising from squatting are used as a test to assess IOH , 7. The The frequency of an abnormally large fall in systolic BP upon active standing from supine in patients with a typical history of severe complaints of IOH occasionally resulting in syncope.The hemodynamic mechanism underlying the abnormal large initial fall in BP upon active standing from supine.The underlying hemodynamic mechanisms of this initial fall in BP upon standing from supine and from squatting.The effectiveness of buttock clenching on IOH using squatting as a provocation.In the present study, we report about our experience of more than 10 years with IOH in teenagers and young adults i.e., age ≤30 years. In a retrospective study we analyzedFrom January 2000 up to August 2011, 1651 patients were referred to the syncope unit in the Academic Medical Centre of the University of Amsterdam for the evaluation of transient loss of consciousness or severe presyncope. Out of the 1651 patients, 371 were aged ≤30 years. A primary clinical diagnosis of IOH was present in 26 of these 371 patients  (7 %).The current analysis focuses on the 26, otherwise healthy, subjects with a primary clinical diagnosis of IOH. These IOH patients had no relevant co-morbidities and did not use vasoactive medications. They all had a history of frequent complaints of severe light-headedness and on occasion syncope in the first 15 s after active standing up.After consulting the medical ethical committee, an approval was not needed for this retrospective study.The test protocol used was identical in all 26 retrospective studies. In order to measure the initial orthostatic BP drop and to test for orthostatic hypotension, the active lying-to-standing test was performed with continuous non-invasive BP monitoring. IOH was defined as a transient decrease in systolic BP of >40 and/or >20 mmHg in diastolic BP within the first 15 s of standing  were available for analysis . Electronic files for computer analysis of the underlying hemodynamics were available in 7/15 patients. The missing The clinical history of these six IOH patients consisted of severe episodes of light-headedness upon standing, with occasionally seeing black spots. Syncope, shortly after standing up, was reported in four patients. In all of them, the episodes of light-headedness occurred after standing up and walking about five steps. In addition to severe complaints of IOH, episodes of vasovagal fainting were reported by 3/6 patients. None of the patients complained about episodes of (near) fainting on arising from squatting.The initial fall in systolic BP during standing up from supine amounted to a median of −52 mmHg . The median time at the nadir was 8.5 s (range 6.6–10.0 s). Within 20 s after standing up from supine, systolic BP had recovered to values around supine control values, with an overshoot in 4/6 patients . Five out of six patients (patients 2–6) reported light-headedness 5–10 s after the onset of standing up.p = 0.13 vs. standing up). The median at the nadir was 8.5 s (range 7.7–9.5 s) (p = 0.41 vs. standing up). Within 20 s after standing up from squatting, systolic BP had not recovered to values around supine control values in 4/6 patients (patients 1–4). Patients 1–3 reported light-headedness 5–10 s after arising standing up from squatting.The initial fall in systolic BP during arising from squatting amounted to a median of −49 mmHg  , whereas in other patients only a small fall in systolic BP was present upon active standing from the squatting position . The peak HR rise at the moment of the nadir upon active standing from supine amounted to 34 bpm (range 26–42 bpm). The peak HR rise at the moment of the nadir upon standing from squat amounted to 28 bpm (range 8–46 bpm).p = 0.56) and in SVR to −31 %  (p = 0.16).The CO and SVR responses at the moment of the BP nadir during active standing from supine showed a large scatter. Both, a large fall in CO and/or a fall in SVR, could underlie this large initial BP dip. At the moment of the nadir, the change in CO amounted to −8 %  (p = 0.03). At the moment of the nadir, the change in CO amounted to +23 %  (p = 0.09) The median changes in CO between supine to standing (−8 %) and squat to stand (+23 %) differed (p = 0.03). In SVR these changes from supine to standing (−31 %) and squat to stand (−42 %) did not differ (p = 0.06). However, the starting value of the SVR in the squatting position was higher than in the supine position (p = 0.03) while CO was comparable (p = 0.22).The hemodynamics underlying arising from the squatting position showed a much more consistent pattern. In the squatting position SVR fell in all six patients to −42 %  after standing up  of the referred patients. An abnormally large fall in systolic BP (≥40 mmHg) was present in only 15/26 (58 %) of these IOH patients.The physiological mechanisms underlying IOH after standing up from supine vary. Both, a large fall in CO and in SVR may occur, whereas after arising from the squatting position a fall in SVR is a consistent underlying mechanism.Effect of buttock clenching is evident. The BP increases markedly by applying this maneuvre.The main findings of the present study are:Current views on diagnostic testing  are of dThe therapeutic aim in young patients with severe symptomatic IOH is functional recovery and improvement of quality of life. If the complaints of transient light-headedness, seeing black spots or even (near-) syncope occur upon active standing, there is hardly a differential diagnosis. Other conditions with “dizziness” and apparent loss of consciousness that may be elicited by standing up, like benign paroxysmal positional vertigo, anxiety, psychogenic pseudosyncope and malingering, have a different presentation. The latter two conditions occur upon active standing, but are in our experience extremely rare (<1/1000 presentations).Considering the lack of malignant causes of syncope for complaints of IOH, we suggest that a typical clinical history of IOH alone suffices to reach to a highly likely (80–100 % certain) diagnosis. In case an abnormal fall in systolic BP (>40 mmHg) is documented in the laboratory accompanied by typical complaints, the diagnosis becomes 100 % certain Fig. . Twelve Four patients with IOH and syncope reported sudden black out and fainting after standing up and walking some five steps. This presentation of symptoms, occurring after walking a few steps, is typical in our experience. However, it has never been studied whether walking a few steps after standing up increases the initial BP fall compared to standing still after arising. The interval between the moment of standing up and the onset of syncope corresponds to the latency between the onset of cerebral hypoperfusion and symptoms, which is approximately 6 s . CerebraPrevious studies in healthy teenagers and young adult subjects using beat-to-beat measurement of SV with calculation of CO and SVR, have established that CO actually increases with the onset of standing up, whereas SVR falls markedly –20. MoreActivation of cardiopulmonary baroreceptors by the elevation in RAP at the onset of whole body exercise and subsequent sympathetic withdrawal.Rapid vasodilation in the contracting leg muscles with the effort of standing.An increase in the A–V pressure gradient .The initial fall in BP upon standing is due to a mismatch of this increase in CO and a decrease in SVR and does not occur, or is far less pronounced, on a passive change of posture , 17. ThrThe first factor is unlikely as the time constant for vascular relaxation is too long. In addition, it has been shown that reflex vasodilation in the forearm was not observed during arising from the squatting position . The secThe initial hemodynamic mechanisms underlying the BP fall upon standing are thought to be similar after supine rest and squatting . This stThis study confirms that the fall in BP after arising from squatting is based primarily on a fall in SVR , 8, 21. The rapid dilatation in leg muscles upon arising from a squatting position can be attributed to the combination of relative ischemia due to compression of blood vessels, active muscle contraction and elevated lower limb arterio-venous pressure gradient , 12, 21.The initial fall in BP after standing from supine and squatting was similar in our study (−52 mmHg vs. −49 mmHg). Earlier studies have shown that the trough of BP after arising from squat is deeper than the trough after standing up from a supine position . The difIn accordance with previous studies, buttock clenching was very effective to abort IOH Fig.  9, 10, , 10, 22.This study has several limitations. The first limitation concerns the small number of study subjects, this as a result of the criteria used for this study. The second limitation is the retrospective nature of this study. As a result, not all suitable files could be used. Also more specific patient details concerning history, complaints and other measurements were not available. Nevertheless, with an experience of more than 10 years of IOH and 1651 syncope patients visiting the syncope unit, this study answers the objectives and puts a new light on the current understanding of the hemodynamics underlying the large initial fall in systolic BP after standing from supine and squatting position. The present study can be considered as a starting point to discuss the role of standing from supine and arising from squatting to diagnose, study and manage the mechanism of IOH.IOH is a frequent cause of syncope and the clinical diagnosis is based on history taking. An abnormally large fall in systolic BP can only be documented in laboratory settings in 58 % of the subjects. For IOH upon standing after supine rest, the hemodynamic mechanism can be either a large fall in CO or in SVR, for IOH upon arising from squatting a large fall in SVR is a consistent finding.


Patients often suffer from physical and mental stress in dental implant surgery. The aim of this prospective study is to investigate the relationship between patient character and blood pressure in dental implant surgery.Fifteen patients were recruited for the present study. All patients had never received implant treatment in the past. To evaluate the patients’ personality trait, NEO-Five Factor Inventory (NEO-FFI) was used. All patients answered 50 questions at the first visit and divided in five dimensions: neuroticism, extraversion, openness, agreeableness, and conscientiousness. The index of physical stress was evaluated by blood pressure and pulse rate.= 0.584, 0.526, p < 0.05). On the other hand, there was no significant correlation between systolic blood pressure elevation and neuroticism score.Ten females and five males (mean 55.5 ± 10.6 years) were evaluated in this study. A significant positive correlation was found between elevation rate of diastolic blood pressure/mean blood pressure and neuroticism score (rs In this limited study, there was significant correlation between neuroticism character and diastolic blood pressure or mean blood pressure rising in patients who received implant surgery. Implant prosthesis has already become one of the treatment options for missing teeth. In addition, under the appropriate maintenance therapy, its longevity is equal or higher compared to the other prosthetic treatments. On the other hand, patients who want to receive the implant treatment cannot avoid the insertion surgery. In addition, if there are hard or soft tissue defects at the planned site, even an additional surgery is necessary , 2. TherIn many cases, we can reduce physical stress under the appropriate anesthesia and monitor general conditions during or just before implant surgery . Mental However, in some patient cases, many dentists experienced the uncontrolled patients even if they provided enough informed consent and appropriately managed the general condition at the surgery; as a result, they had to stop or cancel the implant surgery. One of these reasons is thought to be the influence of the patient’s character. Pavek et al. concluded that mean levels of blood pressure of 85 men during a whole day are slightly affected by particular personality traits . PilqrimFifteen patients were recruited for the present study. All the patients had never received implant treatment in the past. The patients were not accepted into this study if they met any of the following exclusion criteria: (1) experience of implant treatment in the past, (2) general contraindications to implant surgery, (3) moderate or severe hypertension or cardiovascular system disease, and (4) mental disease. All the patients were informed about the purpose of this study and provided their signed informed consent.To evaluate the patients’ personality trait, NEO-Five Factor Inventory (NEO-FFI) was used. All the patients answered 50 questions at the first visit and scored 0–40 in five dimensions: neuroticism, extraversion, openness, agreeableness, and conscientiousness. The index of physical stress was evaluated by blood pressure and pulse rate. All the patients’ blood pressure (systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP)) and pulse rate (PR) were measured by a biological information monitor  at the first visit, entering into the operation room without local anesthesia given before and after the surgery. Elevation rates (ER) of each blood pressure and PR were calculated. Rate pressure product (RPP) was also calculated by SBP and PR.All the patients received implant surgery under local anesthesia , and a one- or two-stage surgical approach was selected according to the initial torque values and resonance frequency analysis (RFA). Screw-type dental implants  were used in all the patients.p < 0.05 was considered statistically significant.SBP_ER, DBP_ER, MBP_ER, PR_ER, and RPP_ER and five dimensions of NEO-FFI were used as correlated variables. For statistical analysis, Spearman’s rank correlation was used and This study was approved by the Ethics Committee of Osaka University Graduate School of Dentistry (H24-E16-1).× mmHg and 143.0 mmHg; SBP, 87.1 mmHg; DBP, 105.7 mmHg; MBP, 69.0 bpm; PR, RPP, 9859 bpm × mmHg just before the surgery. A significant positive correlation was found between DBP_ER/MBP_ER and neuroticism score . There was no significant correlation, but the same tendency was found with extraversion score  in DBP_ER and MBP_ER. On the other hand, there were no significant correlation between SBP_ER, PR_ER, RPP_ER, and neuroticism and extraversion scores. In addition, other personality traits have no correlation with blood pressure, pulse rate, and RPP  were evaluated in this study (Table The blood pressure is considered to be affected by many factors, including physical and psychological stress in dental treatment. From a physical aspect, there are many reports about the relationship between blood pressure and local anesthesia or pain –11.In fact, Tsuchihashi et al. reported that there was a correlation between increased blood pressure and infiltrated anesthesia amount .On the other hand, there are not so many studies about the blood pressure change affected by the psychological aspect.In this study, we found that neuroticism is significantly correlated with DBP and MBP increase. Ploubidis et al. reported that a person with a high degree of neuroticism assessed with the Eysenck Personality Inventory tended to self-select situations likely to lead to adversity and distress . Guasti There were no severe problems during the implant surgery because the patients in this study did not have moderate or severe hypertension or cardiovascular system disease. However, we should pay enough consideration to elder people or patients who have these systemic diseases and neuroticism or extraversion character during implant surgery. Applications such as nitrous oxide or intravenous sedation may be desirable for these patients. In fact, Ichinobe et al. reported that intravenous sedation stabilized measureable changes in blood pressure and pulse rate due to fear and anxiety about dental treatment .On the other hand, there was no correlation between SBP and neuroticism character. For this reason, it is thought that the range of SBP is usually wide compared to DBP. However, there are some reports about the correlation between SBP and patients’ personality trait . BesidesMeasurement of a stress marker such as cortisol or amylase in saliva is one of the useful methods of stress evaluation. Umeanula reported that patients who received tooth extraction had increased cortisol concentration in saliva and concluded that evaluation of a stress marker is a simple, effective method for stress evaluation . HoweverIn this limited study, there was a significant correlation between neuroticism character and diastolic blood pressure or mean blood pressure rising in patients who received implant surgery.


Blood pressure (BP) measurement is a central element in clinical practice. According to international recommendations 3 to 5 minutes of resting is needed before blood pressure measurement. Surprisingly, no study has modelled the time course of BP decrease and the minimum resting-time before BP measurement. A cross-sectional bicentric observational study was performed including outpatients addressed for vascular examination. Using two automatic BP monitors we recorded the blood pressure every minute during 11 consecutive minutes. The data was analyzed by non-linear mixed effect regression. Systolic (SBP) and diastolic BPs were studied and we tested the effect of covariates on its evolution through log-likelihood ratio tests. We included 199 patients (66+/−13years old). SBP was found to decrease exponentially. Simulations based on the final model show that only half the population reaches a stabilized SBP (defined as SBP + 5 mmHg) after 5 min of resting-time while it takes 25 min to ensure 90% of the population has a stabilized SBP. In conclusion, our results and simulations suggest that 5 minutes are not enough to achieve a stabilized SBP in most patients and at least 25 minutes are required. This questions whether the diagnosis of hypertension can be reliably made during routine visits in general practitioners’ offices. Various studies have shown that elevated blood pressure increases cardiovascular diseases such as stroke, myocardial infarction, and peripheral artery disease5. In 2010, high blood pressure was recognized as the major risk factor for global disease burden before tobacco smoking and alcohol use6. Yet, lifestyle modifications  and treatments can reduce and control blood pressure, and thus allowing reducing the morbidity and mortality in specific group population11.Hypertension is a highly prevalent disease worldwide affecting currently more than 1 billion people12. These guidelines suggest a resting time varying from 3 min to at least 5 min before blood pressure measurement12. Surprisingly, no study has modelled the time course of blood pressure decrease and the minimum resting time before blood pressure measurement in a population that should be screened for hypertension.The American Heart Association (AHA), the European Society of Cardiology (ESC), the European Society of Hypertension (ESH) and others have proposed recommendations for blood pressure measurement14. Confirming this hypothesis could have important implication regarding hypertension screening. Indeed, following current guidelines could potentially mean that a significant number of individuals are wrongly diagnosed as hypertensive. This potential overdiagnosis could lead to spending money and giving inappropriate medications thus causing adverse events such as falls.The hypothesis is that the resting time before the blood pressure measurement should be longer than 5 minutes to reach the stabilization of the blood pressure in patientsIn this observational study, we evaluated the time course of the blood pressure decrease during the resting time before the blood pressure measurement in patients addressed at our vascular offices and the parameters explaining the blood pressure decrease during the resting time.Between September 2014 and April 2015, 199 patients were recruited. Table Figure Figure An additional analysis was undertaken to reduce the variability in k by defining a covariate reflecting the shape of the decrease. Individual non-linear regression showed that a non-linear model fit the data best in 128 subjects (64%), while 44 (22%) had a linear decrease and 24 (14%) did not exhibit significant variation of BP. This covariate was included in the model, and was found to impact both the systolic Prest and the time to stabilisation, yielding 3 groups of subjects with different values of k: fast, regular and slow stabilisers (further details in the Appendix). Taking into account individual decrease shape allowed reducing the variability in k by 40%.Simulations using the final model showed that only 50% of the population was stabilised to within 5 mmHg of systolic Prest after 5 min resting time, while up to 25 min may be needed to ensure stable BP in 90% of the population (full results in the Appendix). This resting time falls to 15.0 min if a variability of 10 mmHg is accepted.Figure 5mmHg in 90% of the population is 25 min. Refining the analysis by considering individual variations in the evolution of SBP, we found that there were three subgroups of patients. The regular and fast groups had the same estimated systolic Prest suggesting the “fast” group is in fact stable from the start, while slow stabilisers take much longer to reach a stable SBP than the majority of subjects.Our study showed that among outpatients addressed for a vascular examination the minimal resting time before blood pressure measurement to obtain a stable SBP15. AHA scientific statement stated, “ideally, 5 minutes should elapse before the first reading is taken”12. The ESC have shortened this resting time and suggested a time between 3 to 5 minutes15 whereas “The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure” have increased this resting time writing that “at least five minutes” were required5. Therefore, according to our results, the current leading guidelines about the office measurement of blood pressure might contribute to increase the prevalence of hypertension in our societies, and consequently increasing the number of patients receiving antihypertensive drugs.The resting time found in this study is far longer than the resting time proposed by all previous scientific statementsThe other interesting point is that we found 3 different subgroups of patients with three different patterns of pressure evolution within the resting time. The “fast” pressure group hardly needed to rest for pressure to stabilize. To obtain stable pressure for 90% of the patients in the “regular decrease pressure” group, 9 min were needed whereas up to ~53 min were needed in the “slow decrease pressure” group, although this last figure 2. However, applying our results in clinical practice at the GPs office is not realistic due to time constraints. Indeed, the mean duration of GP consultation was evaluated as 10.7 + /−6.7 minutes in six European countries16 and sixteen minutes in France17. Thus in our opinion hypertension diagnosis should be performed in a dedicated place during a specific consultation. At the very least, a suspicion of hypertension in a patient should warrant a second measurement after a longer rest period than the current recommended 5 min. Another option could be to use an out-of-office blood pressure measurement that is currently devoted to specific clinical conditions such as suspicion of white-coat hypertension or masked hypertension for example18. Even in these cases and according to our results, it seems that patients should be counseled that they have to rest at least 25 minutes and always wait the same time before blood pressure measurements. Our findings corroborate recent result showing that BP measured after a 30 mn rest is significantly lower than BP measured according to the current recommendations19.The other point raised by the study concerns the organization of the public health system to determine where and how can we diagnose hypertension. It is widely admitted that office blood pressure measurement remains the gold standard for screening4.Finally, when we extrapolate our results in France where ~15 million patients are treated for a hypertension we can estimate that 696 000 patients could be over diagnosed for an hypertension. This could represent an annual health public cost of 292 million euros only for the treatment cost. Furthermore giving antihypertensive drugs to patients who do not need it might also provoke drug adverse events19, who showed a significantly lower BP measured after a 30 minutes rest compared to BP measured during a routine consultation. On the other hand, the general characteristics of our population are nearly similar to those included in large randomized controlled trials on hypertension10. Furthermore, our outpatients came for a suspected vascular problem and should have benefited from a blood pressure measurement in order to control this cardiovascular risk factor15. Second, in this study, blood pressure measurements were not compared to home or ambulatory blood pressure measurements because it was not the aim of this study, and home blood pressure or ambulatory measurements are not realized in the majority of patients screened for hypertension. Third, the association of blood pressure values at specific times from baseline with cardiovascular events or target organ damage was not evaluated. As our data showed different models of blood pressure decrease upon time, it would be interesting to perform an additional study to determine which value is the best associated with cardiovascular complications. Fourth, the study included all patients addressed for a vascular appointment, and was thus not designed to test for changes in BP evolution or resting BP in populations with specific features such as comorbidities or treatment. However we did find some differences in the analysis, with older patients in particular having both higher BP and larger differences between baseline and resting BP. The model and estimated parameters from the present study could be used to design future studies investigating specific target populations20. Finally, repeated blood pressure measurements that induce repeated short arterial occlusion might have a role in the blood pressure decrease since transient ischemia during cuff inflation and reactive hyperemia after cuff deflation can induce dilation of upper arm artery, which is likely mediated by baroreceptor reflex and endothelium-dependent vasodilation22. However, these physiological responses are seen after occlusions that last 5 minutes22. When measuring blood pressure with the automatic device, the occlusion lasts only 10 s meaning that for 11 measurements the total duration of occlusion is less than 2 minutes. As we performed repeated occlusions, these can also mimic ischemic preconditioning24. However it has been shown that: i) there was no evidence for ischemic preconditioning during a repeated vessel occlusion that lasts 2 minutes25, and ii) a minimal interval of 2 minutes is needed to observe a significant effect26. Therefore it is not likely that such phenomenon occurs. Furthermore, Nikolic et al. measured the ankle SBP in 250 treated hypertensive patients at 5 and 10 mn (taking the average of two measurements 1 mn apart) and found a decrease of 4 mmHg on average between the two measurements (SD 14 mmHg), which is the same as the decrease we observed in our patients, and suggests that the additional measurements taken in our study do not impact the evolution of SBP too strongly27. As a final note, all international recommendations suggest to perform several measurements of blood pressure and to perform a mean of the measurements15.Our study has several limitations. First, our population is a population of outpatients addressed to a vascular examination and not patients issued from GPs’ offices. Because they came for a regular visit, the observation period was limited to the duration of a normal consultation so we could not obtain measurement after 11 minutes, and some of our findings need to be verified using data collected in a longer study. Interestingly, our results are in accordance to a recent study from Bos and BuisOur study suggests that the current recommended practice of measuring SBP after 5 minutes of resting may not allow for adequate stabilization of SBP, which we find could take at least 25 minutes. Public Health Policies should take into account this result to organize the best way to diagnose hypertension in our societies and avoid overdiagnosis.A cross-sectional, observational, bi-center  study was performed in France. The ethics review board of our institution (named “Comité d’éthique du CHU de Rennes”) approved the “Opti-PA study” . All included subjects signed an informed consent. This study has been conducted according to the principles expressed in the declaration of Helsinki.Outpatients (18 years of age or older), arriving on foot for a vascular examination either for a carotid ultrasound exam or suspected peripheral artery disease, or suspected venous disease or a cardiovascular prevention visit. Only patients in whom arm pressure could be measured in both arms were included. Patients with fistula or lymphedema after breast cancer were not included. Several variables were collected such as age, gender, body mass index, comorbidities and medical treatments during the medical interview or retrieved from medical record.Demographic and clinical data were recorded at inclusion. Each patient was invited to participate in this study when coming into the office room, then the patient was invited to lie down on a bed or to seat on a chair in a controlled room temperature for the blood pressure measurements. Two cuffs adapted to the arm circumference were placed at heart level on each arm of the patient. Heart rate as well as systolic and diastolic blood pressures were automatically and simultaneously measured at each arm every minute for 11 minutes, using two Dinamaps CARESCAPE V100 .The primary study outcome was the blood pressure measurements every minute for 11 minutes.The statistical analysis was performed first on Systolic Blood Pressure (SBP) and second on Diastolic Blood Pressure (DBP). We followed for each subject the SBP or DPB for the arm with the highest measurement at the first measurement 1 mn). The main objective was to model the decrease of blood pressure. The secondary objective was to identify the explanatory variables of this decrease, including the general characteristics of patients described in Table  mn. The Baseline pressure was the first pressure measured after 1 minute of resting. A repeated measure ANOVA analysis was first performed to determine whether a significant trend in time could be detected. The data were then analyzed through non-linear mixed effect regression using the Monolix software. We assumed an exponential decrease of blood pressure P(t) with time t from an initial value to an asymptotic resting pressure (Prest), according to the following equation:5mmHg, which was defined as SBP within 5 mmHg of systolic Prest. We also looked at stable SBP10mmHg (SBP within 10 mmHg). The variability of the automatic blood pressure monitor is expected to be 5 mmHg according to the manufacturer.The model and parameter estimates were used to predict the time when most subjects are expected to reach a stable SBP15. We also report the associated prediction intervals.A patient is considered as having hypertension when measured SBP is equal or above 140 mmHg. To investigate the impact of resting time on the proportion of subjects predicted as hypertensive, we simulated 1000 replicates of the original datasets by sampling parameters from the final model estimates, and computed the corresponding proportion of subjects for various measurements (single measurement or mean of two points 1 or 2 mn apart)The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.Supplementary Information


Longitudinal data are often collected to study the evolution of biomedical markers. The study of the joint evolution of response variables concerning hypertension over time was the aim of this paper. A hospital based retrospective data were collected from September 2014 to August 2015 to identify factors that affect hypertensive. The joint mixed effect model with unstructured covariance was fitted. A total of 172 patients screened for antihypertensive drugs treated were longitudinally considered from Felege Hiwot referral.−4) was significantly best fit to the data. The correlation between the evolutions of DBP and SBP was 0.429 and the evolution of the association between responses over-time was found 0.257. Among all covariates included in joint-mixed-effect-models, sex, residence, related disease and time were statistically significant on evolution of systolic and diastolic blood pressure. The joint modeling of longitudinal bivariate responses is necessary to explore the association between paired response variables like systolic and diastolic blood pressure. Fitting joint model with modern computing method is recommended to address questions for association of the evolutions with better accuracy.The joint mixed effect model with unstructured covariance (AIC: 12,236.9 with  Hypertension is a chronic condition resulting from high blood pressure in the arteries during circulation. Clinically, a person is said to be hypertensive if the individual’s systolic blood pressure (SBP) is greater than 140 mm Hg and or diastolic blood pressure (DBP) is greater than 90 mmHg and those who were already under medication . MonitorWhen several markers are measured repeatedly, joint modeling was used in the context of jointly studying time to clinical event and repeated measures on surrogate outcomes –8. OtherHospital based retrospective studies were conducted among hypertensive patients attending antihypertensive clinic between September 2014 to August 2015 at Felege Hiwot referral hospital, Bahir Dar, Ethiopia. The area is selected because it is central referral hospital that provides organized hypertension follows up care. The sample size was calculated using 95% confidence level and the overall sample size was 172 which after adding 5% for non-response. A systematic random sampling method was adopted for selecting a representative sample from the list of the medical charts that contain the list of hypertensive patients’ name and identification number. Patients were selected randomly using their unique identification number. The study considered all hypertensive patients, whose age was above 15 years regardless of their treatment category during the study period in the referral hospital.In this paper, a longitudinal data on two markers of hypertension (SBP and DBP), various socioeconomic status covariates and clinical factors were considered. SBP and DBP measurements were collected at baseline (SBP0 and DBP0), and every 3 months (SBP3 to SBP12 and DBP3 to DBP12) thereafter. In order to ensure sufficient available information, only a sub-sample of patients having at least four repeated with equally spaced measurements were included in the analyses. Thus, a follow-up data were included hypertensive patients had a maximum of five repeated measures on blood pressure. Data were entered and edited in SPSS v. 21 and analyzed by SAS v. 9.4 and the 95% confidence interval was used to determine significance tests. The missing data for both markers were replaced by last observation carried forward (LOCF) as .ij1 and SBPij2 be the bivariate outcomes for the ith subject measured at jth times for outcomes 1 and 2.Multivariate longitudinal data arise when a set of different responses on the same unit are measured repeatedly over time. A joint modeling of such kind of data is necessary to quantify, firstly, the relationship between evolutions of different responses and, secondly, the evolution of the relationship between different response variables over time. Thus, a pair wise fitting approach has been used in this study as proposed in the literature , 16. Thei sampled measurements of the marker k .We define a general bivariate linear mixed effect model including a random component, a variance–covariance process, and an independent error. Let To take into account correlation between both markers the following bivariate linear mixed effect model was used.The mixed-effect models assume that the regression coefficients are a random sample from some population of the possible coefficient and allow one to model variations between study units . The rani, at each occasion with this model: In this paper, a bivariate linear mixed model including random effects and independent measurement error for both SBP and DBP was presented as outcome variables in each occasion. The two end points were longitudinally measured as a vector of responses, YThe sample was composed of 172 patients with antihypertensive drugs treated, of which 38 (22.09%) and 21 (12.51%) patients had diabetic and stroke and 76 (44.19%) patients had no other related disease. The results showed that, 93 (54.09%) women, with a mean age of 51.87 (SD = 14.33) years. Whereas mean age of male patients was 56.82 (SD = 14.98) years and the mean systolic and diastolic blood pressure of patients declined from baseline to the next 3 months period, to the next 6 months period and soon up to the last 12 months period follow up time. The baseline SBP mean of patients was 153.58 (SD = 31.60) mmHg and declined to 129.48 (SD = 20.07) mmHg over time and similar history was found in DBO patients. These summaries suggest that the treatment has a significant effect on systolic and diastolic blood pressure over the follow-up time , 3 months period (P  = 0.001) and 6 months period (P  = 0.011) systolic and diastolic blood pressure were significantly different compared with 12 months period follow up time. The systolic and diastolic blood pressure of rural patients (P  = 0.0308) was significantly different compared with urban patients. The systolic and diastolic blood pressure patients with diabetic showed a significant difference at baseline (P  = 0.0077) and 3 months period (P  = 0.0015) compared to patients with stroke (Table −4) was significantly best fit to the covariates. This results in line with [The joint mixed effect model with unstructured covariance  between the baseline and after a 12-month follow-up-time. This result in line with –23 as thThe joint mixed effect model with unstructured covariance was preferred among others to fit the data. It can be generalized that, the two outcomes have a strong positive correlation and the joint mixed effect model was preferred. Thus, joint modeling of longitudinal bivariate responses is necessary to explore the association between paired response variables. The baseline mean of the two symptoms was out of the normal range for hypertensive patients but it declines through clinical treatment follow-up time of 3 month period intervals. As the joint model is able to address questions for the association of the evolutions with better accuracy, fitting joint mixed effect model is recommended.The joint modeling problem is failing to convergence because of a large number of parameter estimates. Moreover, some one also might want to look at modeling more than two response variables over time. Thus one might be able to be implemented using modern computing method for future work to model more than two response variables over time. Here in the joint mixed effect model, authors did not see the interaction effect of the predictors over time. Therefore, researchers should consider the contribution of the interaction effect of the predictors on to the joint mixed effect model.


N = 153) is very high (11.7 U/gHb) compared to healthy subjects (N = 80) ( 5.6 U/gHb). Lower values were observed in transplant patients with kidney from living donors (N = 16) (9.8 U/gHb). Except for steroids, no correlation has been found with the immunosuppressive therapies and routine clinical and laboratory parameters. Also serum oxidized albumin, which reveals oxidative stress, is significantly higher in transplant patients from cadaver donors (53%) compared to that from living donors (36%). Overall, these data indicate that most of transplant kidneys from cadavers lost part of the detoxifying power against circulating toxins and suffer a relevant oxidative stress compared to those coming from living donors. A case report suggests that e-GST could represent a very early marker of incipient graft rejection. In conclusion, e-GST may be used to check the decline or maintenance of the kidney detoxification competence during post-transplantation course.Erythrocyte glutathione transferase (e-GST) is overexpressed in case of increased blood toxicity and its level correlates with the kidney disease progression. Thus, it represents a probe of kidney efficiency against circulating toxins. We measured the activity of e-GST in patients with transplant kidney from living and cadaver donors, correlated its level to biochemical parameters of kidney function, and measured the level of oxidized albumin as a probe of oxidative stress using a new simple procedure. Interestingly, the activity of e-GST in transplant patients from cadaver donors ( KT improves the quality of life and decreases the cardiovascular mortality and morbidity, compared to dialysis5. For example, about 20 years ago a large study involving 220,000 patients receiving long-term dialysis or cadaveric transplant demonstrated larger benefits about mortality in transplant patients after 18 months from the graft1. Also, a more recent study showed that KT is the most safe method among renal replacement therapies in patients with polycystic kidney disease because it is associated with a low morbidity and a high patient survival rate6. These results have also been highlighted even in patients older than 70 years; in fact the transplantation in these patients offers survival advantages over dialysis treatment7. New advances in immunosuppressive therapy induced a decrease in cases of acute rejection and improved graft survival9. An accurate correlation between immunosuppressive dosage and the outcome of transplant kidney is required in order to provide adequate pharmacological treatment 9. The imprecision and inaccuracy in the predictiveness of diagnostic biochemical markers currently in use, makes pharmacological treatment more difficult10. To date, no biomarkers able to assess the ability of the transplanted kidney in removing many different toxic compounds are available. The present study focuses on a first application of erythrocyte glutathione transferase (e-GST) as a useful tool to check the competence of transplanted kidneys in this specific role. e-GST (synonym of human glutathione transferase P1-1 (GSTP1-1)) is an homo-dimeric intracellular enzyme of about 46 kDa that represents the most abundant form of intra-erythrocyte transferase 11. More in general, glutathione transferases (GSTs) are a superfamily of iso-enzymes able to promote the conjugation of glutathione with many toxic compounds of different chemical nature and shapes, and thus favoring their excretion12. Human cytosolic GSTs have been grouped into seven gene independent classes . In addition, GSTs may act like ligandins by sequestering many small and/or large toxic compounds and peptides. Furthermore, GSTP1-1 appears to control signaling pathways and transcriptional responses of cells through protein–protein interaction mechanisms13. GSTs are also able to bind the dinitrosyl-diglutathionyl–iron complex, a natural toxic derivative of nitric oxide, which becomes harmless when bound to GST14. Besides classical transcriptional activators , GSTP gene is modulated by the levels of endogenous activators. These include toxins produced by the functional defect of depurative organs such as liver and kidney15. In this context, e-GST, compartmentalized in the erythrocytes and not dialyzable, seems to be an interesting long-term biomarker either to assess kidney function and also the adequacy of dialytic techniques16. e-GST assay represents a very simple, inexpensive, and fast laboratory tool in clinical nephrology also developed for high-throughput routine protocols17. e-GST may be considered a sort of ideal long-term biomarker that should provide a measure of circulating toxins in temporal spans not limited to a single day or dialysis session but extended up to one/two months16. The e-GST assay, specific and accurate for this enzyme18, has been applied in several studies concerning validation and use of e-GST as biomarker in hemodialysis patients17 and in the assessment of dose and adequacy of dialysis therapy19. Moreover, e-GST was found to be overexpressed in chronic kidney disease (CKD) patients under conservative therapy20 and in type-2 diabetes mellitus patients (T2DM) with CKD22. Finally, environmental23 and endogenous factors like autoimmune disease24 also affected e-GST levels in healthy subjects and in non-uremic patients. In this study, we use e-GST like a probe able to assess functionality of transplant kidneys coming from living and cadavers donors in their specific action to scavenge endo and exogenous toxins. In addition, we also adopted a very simple and inexpensive procedure to evaluate the amount of oxidized albumin (HSAox) in serum of transplant patients. This protein shows 17 disulfide bridges and only one reduced cysteine (Cys34). This residue can react with serum disulfides to give glutathionylated or cystationylated protein. A few amount of this residue may also give higher oxidized forms such as sulphinic and sulfonic derivatives. This oxidized protein represents a reliable short-term biomarker of oxidative stress related to pathological conditions27. Interestingly, HSA is considered the major plasma protein target of oxidative stress in uremia28. In fact, relevant increase of this oxidized protein has been found in hemodialysis patients29, in CKD patients under conservative therapy30 but never evaluated in kidney transplant patients. Our data indicate that both e-GST and HSAox are higher than those found in healthy subjects, in particular in kidney transplant patients from cadavers. The levels of these two biomarkers suggest that all transplant kidney both from living and cadaver donors loose part of their detoxifying ability against circulating toxins.Kidney transplantation (KT) represents the treatment of choice in end-stage renal disease (ESRD) patientsThe number of patients enrolled in the present study were: 153 kidney transplants from cadaver donors and 16 kidney transplants from living donors. The epidemiological features were summarized in Table Hb), nephropathic patients under conservative therapy (stage IV according to KDIGO guidelines) (10.7 ± 1.3 U/gHb), and hemodialysis patients under diffusive (10.3 ± 1.1 U/gHb) and convective techniques (8.6 ± 1.2 U/gHb)21  are close to those found in patients with maintenance hemodialysis with diffusive technique or in CKD patients (stage IV)  are higher when compared to other kidney pathology recipients  compared to men (11.1 ± 0.5 U/gHb)  levels of kidney transplant patients (from living and cadaver donors as well) were very similar in all other categories examined , maintenance hemodialysis patients under convective and diffusive techniques)  after 2 months. Values before and after 2 months from transplantation were in the same range of e-GST levels compared to those measured for CKD IV stage (10.7 ± 1.3 U/gHb), hemodialysis patients under diffusive technique (10.3 ± 1.1 U/gHb). The value determined 1 month after transplantation was similar to that derived from transplant patients from deceased donors (11.7 ± 0.4 U/gHb).e-GST level was determined in 153 kidney transplant patients from cadaver and 16 kidney transplant patients from living donors, at least 3 months from the transplantation. The results shown in the present study were compared with data of healthy control group (5.6 ± 0.6 U/g)21 Fig. . The valIV) Fig. . Moreovents Fig. . The levHb) Fig. , as alsoes) Fig. . Furtheres) Fig. . This laes) Fig. . Notably31, even if this marker does not discriminate between toxic and non-toxic compounds. For assessment of eGFR, based on serum creatinine, it is recommended to use the CKD epidemiology collaboration (CKD-EPI 2009) equation33. The level of eGFR in healthy subjects is estimated to be ≥ 90 ml/min. According to Kidney Disease Quality Outcome Initiative (K-DOQI)34 and the Kidney Disease Improving Global Outcomes (K-DIGO)35 the diagnosis of CKD is formulated when eGFR is < 60 ml/min for >3 months with or without kidney disease or when kidney damage is defined by structural or functional abnormalities for >3 months with or without decrease of eGFR. The cutoff of 60 ml/min for eGFR has been chosen because this value is considered an early signal of renal disease and also the lower values are associated with high risk of cardiovascular mortality and morbidity36. For these considerations, we divided all transplant patients into two subgroups based on the eGFR values with a cutoff of 60 ml/min. A remarkable increase of e-GST level is showed in the patient recipient with eGFR < 60 ml/min . The value of e-GST for patients with eGFR ≥ 60 ml/min is lower (9.1 ± 0.4 U/gHb) than e-GST measured in patients with eGFR < 60 ml/min .The eGFR represents up to now the best index for kidney function CKD-EPI 009 equatmin Fig. . Moreovemin Fig.  but a unTable Hb vs. 10.9 ± 0.5 U/gHb, P = 0.0254)  itself, but by the increase of hematic toxicity probably derived from the glyco-metabolic dysfunctions observed in patients in therapy with steroids. However, the patients withdrawn from steroid have an optimal kidney function and have a low risk of rejection.Adequate immunosuppressive therapies are usually administered during post-transplant phase to avoid acute rejection and early graft loss. This therapy is usually constituted by the association of two or three drugs with different mechanism of action. Furthermore, each class of drugs is characterized by a different profile of toxicity. The choice of immunosuppressive treatment is based on both the recipient and donor clinical features. In this context, the possible correlations among immunosuppressive therapies and e-GST levels were studied. No evident correlations were found for e-GST levels and all immunosuppressive “drug combination” dispensed to the patients Fig. , althoug54) Fig. . It has Interestingly, an evident increase of e-GST level has been observed close to an acute rejection phase. During the present study, the value of e-GST was constantly monitored for around 5 months in eight transplant patients from cadavers. While slight increased values with time have been observed for seven samples Fig.  mean in. The rejThe oxidized albumin, used as a short-term biomarker of oxidative stress, suggested the occurrence of relevant oxidative events in the transplant patients, particularly in those receiving kidneys from cadaveric donors (53.4 ± 12.0%)  in 20.2% of transplant patients from cadavers and 0% from living donors  and the donors (such as age and duration of both cold and warm ischemia)40. During renal reperfusion-ischemia, oxidative stress and inflammatory process have been demonstrated. In particular, kidney tissue injury during ischemia/reperfusion comes as a result of membrane lipids peroxidation, oxidative damage of proteins and DNA, and it results in apoptosis and necrosis41. Previous studies have been developed considering the cold ischemia time like a predictor of cardiovascular, nephro-urologic, and endocrine complications42. The observed increase of e-GST activity is an indication of paramount relevance as most of these kidneys display normal morphological structure and thus e-GST may represents an innovative and very sensitive biomarker of kidney efficiency. Overall, all these results may suggest that the better life expectancy found in transplant patients compared to dialyzed patients cannot be due to a lower level of circulating toxins or oxidizing compounds but, more likely, to the absence of frequent cardiovascular stress due to chronic dialytic therapy. In a second experimental section, we monitored eight patients in maintenance dialysis therapy, before KT, 1 and 2 months after transplantation , virus hepatitis B and C, serum alanine aminotransferase and/or aspartate aminotransferase twice the upper limit of normal values, morbid obesity, rheumatologic disorders . Healthy subjects for the determination of HSAox and e-GST were 80 and 40 volunteers, respectively, from the transfusion medicine section of the Policlinico, University of Rome, Tor Vergata.The protocol of this study followed the declaration of Helsinki and all patients healthy subjects provided a written informed consent. The present study was approved by the Ethical Committee of Azienda Ospedaliera Universitaria Policlinico Tor Vergata. A total of 169 patients were enrolled. Inclusion criteria were: age >18 years and renal transplant patients for at least 3 months. Early morning blood samples were taken from each patient for biochemical screening test after 12 h overnight fasting. Blood samples were collected into tubes  containing ethylenediaminetetraacetate (EDTA). Blood was obtained via venipuncture from the antecubital vein. All samples were placed on ice and plasma was separated by centrifugation at 1600 × An automated hematology analyzer XE-2100  was used for the determination of Hemoglobin (Hb). All routine parameters were determined using Dimension VISTA 1500 . The lipid profile , triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol) was determined by standard enzymatic colorimetric techniques . All other parameters were analyzed according to standard procedures in the Clinical Chemical Laboratories of the Policlinico, University of Rome, Tor Vergata.32.The eGFR was calculated using the CKD epidemiology collaboration (CKD-EPI) formula2O2, cystamine, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Ellman’s reagent) and all other reagents were purchased from Sigma–Aldrich .Glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), ethylenediaminetetraacetic acid, H48. Results were reported as enzyme units (U) per gram of hemoglobin (Hb) (U/gHb): one unit is the amount of enzyme that catalyzes the conjugation of 1 micromole of GSH to CDNB in 1 min at 37 °C17.e-GST activity was obtained using a spectrophotometric procedure at 340 nm (37 °C), using an Uvikon 941 Plus spectrophotometer . One volume (40 μl) of whole blood was diluted into 1 ml of bi-distilled water causing fast erythrocytes hemolysis. Then, 100 µl were diluted to a final volume of 1 ml containing 1 mM GSH, 1 mM CDNB in 0.1 M potassium phosphate buffer, pH 6.5 according to Habig and coworkers2O2 1 M according to Beers and Sizer49. Results were expressed as enzyme units (U) per gram of Hb (U/gHb): one unit is the amount of enzyme that catalyzes the decomposition of 1 micromole of H2O2 in 1 min at 25 °C.e-CAT activity was determined with a spectrophotometric assay at 240 nm (25 °C) (Kontron Uvikon 941 Plus). Briefly, 5 μl of hemolyzed blood was diluted in 1 ml of buffer potassium phosphate 0.05 M pH 7.0 with EDTA 0.1 mM, and 10 μl of H50 based on the fast reaction of cystamine with Cys34. The released cysteamine is stoichiometric with Cys34 and easily determined with DTNB (ε412 nm of TNB- : 14.1 mM-1 cm-1). The assay was carried on a Kontron Uvikon 941 Plus spectrophotometer (Kontron Instruments) at 412 nm (25 °C). One volume of 50 μl of serum was diluted in 0.89 ml of potassium phosphate buffer 0.1 M pH 8.0 recording an autozero sample. Suddenly, 50 μl of Ellman’s reagent  and 10 μl of cystamine  were added to the solution and after 15 min of incubation the absorbance was registered.The HSAox was determined by subtracting the value of reduced HSA from the total HSA estimated from the routine clinical assay. The level of reduced HSA cannot be evaluated by direct titration of its reduced Cys34 by DTNB, because this thiol reagent reacts very slowly with this cysteine. We therefore used a modified proceduret-test and Mann–Whitney). The minimal level of significance of the differences was fixed at P < 0.05. One-way ANOVA was employed to compare the data between different data set of transplant patients (from living and cadaver donors). Comparison among groups was performed with the univariate ANOVA with a covariate for continuous parametric variables. Analysis of correlation was done with Pearson coefficient. This analysis was performed using the Statistical Package for the Social Sciences Windows, version 15.0 . The graphic and results visualization were obtained by GraphPad Prism .Data are reported as means ± standard error of the mean (SEM). All continuous variables were checked for normality using Kolmogorov–Smirnov test. Differences between baselines and final values were tested (paired


Background. The assessment of blood pressure (BP) response during exercise test is an important diagnostic instrument in cardiovascular system evaluation. The study aim was to determine normal values of BP response to submaximal, multistage exercise test in healthy adults with regard to their age, gender, and workload. Materials and Methods. The study was conducted in randomly selected normotensive subjects (n = 1015), 512 females and 498 males, aged 18–64 years (mean age 42.1 ± 12.7 years) divided into five age groups. All subjects were clinically healthy with no chronic diseases diagnosed. Exercise stress tests were performed using Monark bicycle ergometer until a minimum of 85% of physical capacity was reached. BP was measured at rest and at peak of each exercise test stage. Results. The relations between BP, age, and workload during exercise test were determined by linear regression analysis and can be illustrated by the equations: systolic BP (mmHg) = 0.346 × load (W) + 135.76 for males and systolic BP (mmHg) = 0.103 × load (W) + 155.72 for females. Conclusions. Systolic BP increases significantly and proportionally to workload increase during exercise test in healthy adults. The relation can be described by linear equation which can be useful in diagnostics of cardiovascular diseases.  Arterial hypertension is a serious medical, social, and economic problem in Poland. In WOBASZ Study conducted in Central Poland, hypertension was identified in 40% of males and 32% of females whereas more females than males were undergoing antihypertensive treatment  –8. AccorThe relationship between the systolic blood pressure values and the exercise workload had already been determined with the linear regression by Heck et al.; however, the correlation was identified for the group composed mainly of athletes (90%) aged 10–71 years [It has already been established that the excessive increase in blood pressure during exercise test on a cycle ergometer or a treadmill can be a prognostic factor for future development of hypertension or death due to cardiovascular complications , 11, 12.The aim of the study was to define normal blood pressure response to multistage exercise test in healthy adults with reference to their age and gender at each workload stage.n = 498) and 50.9% of females (n = 517). The study analysis included the subjects with an age range of 18–64 years (mean age 42.1 ± 12.7 years) divided into the following five age groups: 18–24 years (n = 147), 25–34 years (n = 154), 35–44 years (n = 186), 45–54 years (n = 330), and 55–64 years (n = 198).Randomly selected 2500 people were invited to take part in the study. 1121 of them took part and 106 were disqualified from the study. Eventually, the study included 1015 clinically healthy subjects. Adult participants of the study were randomly recruited based on the information provided by the Local Data Bank in Lodz that stores the data of 760 000 inhabitants. The distribution pattern of the draw was maintained. The attendance rate amounted to 44.8%. The study group was composed of 49.1% of males  in history were excluded from the study. The arterial blood pressure values <140/90 mmHg were considered as normal. The subjects were qualified for the exercise test after medical examination, resting arterial blood pressure measurement, echocardiography at rest, and anthropometric measurements taken . The exercise tests were performed using the Monark bicycle ergometer Ergomedic 874 E where the study subjects were pedaling at 60 revolutions/minute with the workload increased every 3 minutes. Simultaneously, the heart bioelectrical function was being monitored by means of the cardiologic software Marquette CASE-16 and blood pressure was measured by Korotkov method. The arterial blood pressure measurements were performed at rest, at the left upper arm, and after 15 minutes' rest as well as in the last 45 seconds of each workload of exercise test while the subjects were still pedaling. The measurements were taken using the aneroid sphygmomanometer INCO-VERITAS S.A. which was calibrated and checked according to the recommendations of the European Society of Hypertension , 15, 16.x) and standard deviation (SD). A p value less than 0.05 was considered statistically significant. Linear relationships (first-order regression equations) with regard to one standard deviation were determined. ANOVA test and Kruskal-Wallis test were used for statistical analysis.Statistical analysis was performed with Statistica software (10.0 Star Soft). Values are expressed as arithmetic mean (p = 0.1). The characteristics of the study subjects analyzed at rest as a whole group and according to the gender are presented in p < 0.001). Females, in turn, showed a significantly higher resting heart rate in comparison to males (p < 0.00003)  .p = 0.001) for the systolic pressure and by 2.7 ± 1.7 mmHg (p = 0.0001) for the diastolic pressure compared with females at the same age. Systolic and diastolic blood pressure increased statistically significantly with age of the study females and males (p = 0.0001) ( 0.0001) .p = 0.0001) by mean 8.1 ± 1.3 mmHg. Exercise diastolic blood pressure values were statistically significantly higher in males than in females (p = 0.0001). The value of double product (peak HR·SBP) at peak workload of the exercise test was significantly higher in comparison with female subjects (p = 0.00001).The increase of mean systolic blood pressure amounted to 44.4 ± 16.2 mmHg which was equal to 28% of the resting value. Female subjects completed their exercise test at the mean workload of 87.4 ± 36.9 (W) with the heart rate values of 159.2 ± 16.1 bpm, systolic arterial blood pressure 162.4 ± 25.6, and diastolic blood pressure 87.8 ± 11.1 mmHg, whereas males finished their exercise test at the mean workload of 122.2 ± 57.7 (W) with the heart rate values of 163.5 ± 13.2 bpm, systolic arterial blood pressure 173.7 ± 28.8, and diastolic blood pressure 89.8 ± 12.1 mmHg. Compared with the resting values, the diastolic arterial blood pressure during the exercise test increased statistically significantly (x). All the relationships were statistically significant (r from 0.4 to 0.6) (p < 0.0001).In the present study, during single-bout, multistage exercise test on a cycle ergometer, systolic blood pressure increased significantly with the workload both in clinically healthy males and females. The relation between systolic blood pressure and workload can be expressed depending on sex, by the following formulae:For the studied males, sessment , 17, 18.sessment . Clinicasessment .Most studies where diagnostic and prognostic value of blood pressure measurements during exercise test were assessed were carried out on a cycle ergometer. Applying the cycle ergometer enables standardization of the procedure according to the test duration and workload determination, as in treadmill exercise testing. Some authors, however, suggest that the cycle ergometer also enables more accurate blood pressure assessment due to a better shoulder stabilization during measurements . It has Nearly 30 years ago Heck et al. studied the relation between workload and systolic blood pressure in about 3000 healthy subjects aged between 10 and 71 years, 90% of whom were sportsmen [According to Heck, the calculated systolic blood pressure value for a given age and workload should correspond to the mean value increased or reduced by 1SD for the study population . The forFletcher et al. reported the results of a similar study that evaluated the blood pressure response to various workloads . Their fr-Pearson test and test for two regression coefficients. The dependence can be described as a linear relationship. The slope angle between the line reflecting systolic blood pressure values during exercise and the axis corresponding to workload values during exercise was larger in males than in females. The comparison of regression coefficients did not reveal statistically significant differences between systolic blood pressure values during exercise in the analyzed age groups. Systolic pressure increases with dynamic workload as a result of increased stroke volume, whereas diastolic blood pressure usually remains unchanged [In the present study peak values of the systolic blood pressure during multistage exercise test did not exceed 190 ± 20 mmHg in women and 210 ± 20 mmHg in men , 21, 22.nchanged . An excenchanged , 21, whenchanged , 21.The presented ranges of blood pressure values during exercise testing might be useful in clinical practice while diagnosing hypertension and they could facilitate the identification of patients with the hypertensive response to exercise –29. The (1)The relationship between systolic arterial blood pressure and the workload during exercise test in adults can be described by the following equations: For adult males aged 18–64 years, For adult females aged 18–64 years,(2)The values can be applied for monitoring normal and hypertensive response to exercise, particularly in prevention of cardiovascular diseases.