The anti-hypertensive effects of an oral daily dose (15 mg/kg) of the synthesized compounds in spontaneously hypertensive (SHR) rats and normotensive Wistar Kyoto (WKY) rats were analysed. The data demonstrate that the twin drug VB3-QTME both reduces the elevated blood pressure and prolongs the action time in SHR rats without effect on WKY rats. However, definitive evidence of a precise mechanism of action by which VB3-QTME might decrease blood pressure remains elusive. Based on the results, the therapeutic potential of this twin drug is discussed.A novel twin drug consisting of nicotinic acid (VB3) and quercetin tetramethyl ether (QTME) has been synthesized as an antihypertensive in a total yield of 79.2% through methylation, hydrolysis, acylation and esterification starting from rutin. The structures of synthesized compounds were elucidated by  Hypertension is a chronic medical condition that can cause diverse complications which may not only markedly impair the quality of life but also cause a heavy financial burden on families and society. Currently, the incidence of hypertension is the highest in the world among all diseases, affecting one-third of the global adult population . As an iWestern medicines show rapid and remarkable effects against hypertension, but these drugs show toxic effects clinically. Although Traditional Chinese medicine (TCM) has gained much attention clinically because of its advantages of lower toxicity and fewer side effects, protection of target organs, multiple targets, and multiple pathways, its disadvantage is that these drugs require a long time to exert their effects ,9,10. ThEpidemiological studies have shown an inverse association of flavonoid-rich diet consumption with the risk of hypertension and cardiovascular disease . QuercetIn the present study, quercetin was selected for conjugation with nicotinic acid to obtain a quercetin-antihypertensive double prodrug. Although, linking of quercetin with VB3 in 1:1 ratio is difficult due to the presence of a number of hydroxyl groups, we have been able to conjugate this agent in the form of its derivative, quercetin tetramethyl ether (QTME) with VB3. In this paper, synthesis and anti-hypertensive activity of the twin drug of nicotinic acid and quercetin tetramethyl ether (VB3-QTME) are reported.2), the glycoside of quercetin was treated with methyl iodide (CH3I) in dry dimethyl formamide (DMF) in the presence of potassium carbonate (K2CO3). The methylated glycoside 3 was obtained as semisolid, which was subjected to hydrolysis by refluxing in acidic aqueous solution to obtain the quercetin derivative, quercetin tetramethyl ether . The free hydroxyl group generated at 3-position in this derivative was used as a synthetic handle for conjugation with the VB3 (5). Compound 5 was treated with thionyl chloride (SOCl2) by stirring at reflux and after removing excess of SOCl2, nicotinoyl chloride hydrochloride 6 was obtained as a white solid. QTME was treated with compound 6, dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) using dichloromethane as solvent and the desired derivative, VB3-QTME (1) was obtained as light yellow solid, with a total yield of 79.2%. The sequence of various steps involved in the reactions is shown in To conjugate the polyphenolic flavonoid quercetin with carboxyl group containing anti-antihypertensive drugs in 1:1 ratio, an alternative strategy was developed. For this purpose, rutin  was used as the internal standard. High resolution (HR)-electrospray ionization (ESI)-mass spectroscopy (MS) was measured on a Bruker APEX IV FT-MS (7.0 T) mass spectrometer in positive-ion mode. ESI-MS was obtained on an Agilent XCT 6320 IT mass spectrometer.All commercial reagents and solvents were used as received without further purification unless specified and reactions were monitored by thin-layer chromatography (TLC) on normal-phase silica gel GF254 plates. Spots on the TLC plates were visualized using ultraviolet light (254 nm or 365 nm). Silica gel (200–300 mesh) was used as stationary phase to isolate the compounds. 2, 1.8 g, 3.0 mmol) in dry N,N-dimethylformamide (30 mL), anhydrous potassium carbonate  and methyl iodide  were added and the reaction mixture was refluxed for 48 h. The solution was filtered and insoluble potassium salts were washed with acetone. The washings were combined with the filtrate and solvent was removed under reduced pressure to obtain methylated glycoside 3 as a semisolid residue.To a fine suspension of rutin , yellow solid, mp 195–197 °C  δ: 6.33 , 6.53 , 7.53 , 7.72 , 7.79 , 9.47 , 3.87–3.97 . 13C-NMR (DMSO-6d) δ: 172.658 (C-4), 164.147 (C-9), 160.528 (C-7), 156.572 (C-2), 150.338 (C-5), 146.608 (C-4'), 142.148 (C-3'), 138.030 (C-3), 123.705 (C-1'), 120.666 (C-6'), 110.995 (C-2'), 110.341 (C-5'), 106.302 (C-1'), 96.198 (C-6), 95.555 (C-8), 56.581, 56.086, 55.831, 55.627 (4-OCH3). Anal. calcd. for C19H18O7: C, 63.68; H, 5.06; O, 31.25. Found: C, 63.81; H, 5.11. HRMS (ESI) m/z: 359.11804 [M+H]+, calcd. for C19H19O7 359.11253. Spectroscopic data of the compound was consistent with those in the literatures +, calcd. for C25H22NO8 464.13400.QTME  and nicotinoyl chloride hydrochloride  were dissolved in dichloromethane (30 mL) containing DCC  and DMAP . The reaction mixture was stirred at reflux for 12 h. After reaction, the solvent was removed under reduced pressure. After washing with saturated NaSixteen-week-old, male SHR and WKY rats were obtained from Weitong-Lihua Experimental Animal Technical Co., LTD . All the experiments were performed in accordance with Institutional Guidelines for the ethical care of animals. All rats were maintained three per cage at a constant temperature (24 + 1 °C), with a 12 h dark/light cycle and on standard rat chow. An adaptation period of 2 weeks for vehicle administration and blood pressure measurements was allowed before the initiation of the experimental protocols.Twenty-four SHR (six rats/group) were randomly assigned to a VB3-QTME group , a quercetin group , a nicotinic acid+QTME group , a SHR vehicle group(1 mL of 0.5% carboxymethylcellulose sodium). Six WKY were assigned to a vehicle group (1 mL of 0.5% carboxymethylcellulose sodium), another six WKY were assigned to a VB3-QTME group . During the experimental periods all the rats had free access to tap water and chow.Before treatment, rats were trained to the procedure and measurements were recorded. Then rats were dosed once daily by oral gavage with 32.4 mmol/kg of test compounds on each of the 7 days. Systolic blood pressures were measured 4 hours after the oral gavage on each of the 7 days in awake rats by the tail-cuff method . At least six determinations were made in every session and the mean of the middle four values was taken as the systolic blood pressure level. At the end of the last day’s treatment, tail systolic blood pressures were then continuously measured in conscious rats in the following 4 h, 8 h, 12 h, 24 h, 48 h, 72 h.p values < 0.05 were considered significant (SPSS (PASW)17.0). Systolic blood pressures of SHR and Wistar rats before and after treatment are presented on The data were given as means ± SEM. Comparison between experimental groups were made by using one-way ANOVA, followed by student Newman Keuls test. §§§p < 0.001). ◆ represents the changes in tail systolic blood pressures of SHR with quercetin(32.4mmol/kg), and it was observed that the blood pressure declined after 7 days’ administration (ϕp < 0.05). ■ represents the changes in tail systolic blood pressures of SHR orally administered VB3-QTME (32.4mmol/kg), and it was observed that the blood pressure remarkably declined after administration (*** p < 0.001). However, the VB3-QTME group exhibited more significant  reduction effects on blood pressure.p < 0.001). ● represents the changes in tail systolic blood pressures of SHR with nicotinic acid+QTME, it was observed that the maximal reduction of blood pressure at the fourh hour after the 7 days’ administration, and the anti-hypertensive activity of VB3-QTME can last 24 h (*** p < 0.001). However, the VB3-QTME group exhibited longer duration effects on blood pressure.Herein we demonstrated that treatment with nicotinic acid+QTME for 7 days  produced an around 15%-reduction in systolic blood pressure, and the treatment with VB3-QTME for 7 days (15.0 mg/kg/day) almost normalized systolic blood pressure. Similar to our data, other study in SHR documented that treatment with quercetin for 7 days (10.0 mg/kg/day) produced a 10%-reduction in mean blood pressure . AnotherEvidence exists to support several potential mechanisms whereby quercetin might decrease blood pressure and decrease the severity of hypertension in animals and humans. These mechanisms are a decrease in oxidative stress, interference with the renin-angiotensin-aldosterone system (RAAS), and/or improving vascular function in an endothelium-dependent or -independent manner ,43,44,45Herein, an efficient and convenient synthesis of VB3-QTME consisting of VB3 and QTME has been achieved by a four-step reaction starting from the readily available nicotinic acid and rutin through methylation, hydrolysis, acylation and esterification in 79.2% yields is presented. The anti-hypertensive activity of VB3-QTME was recorded, and the data demonstrate that this twin drug both reduces the elevated blood pressure and prolongs the action time in SHR rats without effect on WKY rats. Based on the results, the exact mechanism of VB3-QTME and its applications are yet to be explored further.


To prospectively validate the Predicting Out-of-OFfice Blood Pressure (PROOF-BP) algorithm to triage patients with suspected high blood pressure for ambulatory blood pressure monitoring (ABPM) in routine clinical practice.Prospective observational cohort study.10 primary care practices and one hospital in the UK.887 consecutive patients aged 18 years or more referred for ABPM in routine clinical practice. All underwent ABPM and had the PROOF-BP applied.The main outcome was the proportion of participants whose hypertensive status was correctly classified using the triaging strategy compared with the reference standard of daytime ABPM. Secondary outcomes were the sensitivity, specificity, and area under the receiver operator characteristic curve (AUROC) for detecting hypertension.The mean age of participants was 52.8 (16.2) years. The triaging strategy correctly classified hypertensive status in 801 of the 887 participants  and had a sensitivity of 97%  and specificity of 76%  for hypertension. The AUROC was 0.86 . Use of triaging, rather than uniform referral for ABPM in routine practice, would have resulted in 435 patients  being referred for ABPM and the remainder managed on the basis of their clinic measurements. Of these, 69  would have received treatment deemed unnecessary had they received ABPM.In a population of patients referred for ABPM, this new triaging approach accurately classified hypertensive status for most, with half the utilisation of ABPM compared with usual care. This triaging strategy can therefore be recommended for diagnosis or management of hypertension in patients where ABPM is being considered, particularly in settings with limited resources. Hypertension is an important risk factor for cardiovascular disease,Current strategies for the diagnosis of hypertension recommended by the UK National Institute for Health and Care Excellence and the American College of Cardiology/American Heart Association Task Force state that to confirm a diagnosis of hypertension people should undergo out-of-office measurement (ambulatory or home blood pressure monitoring) if blood pressure is raised in the clinic.Triaging patients for ABPM has been suggested to improve the management of hypertension by limiting the use of ABPM to those most likely to be misclassified by clinic blood pressure measurements.This approach, using the algorithm Predicting Out-of-OFfice Blood Pressure (PROOF-BP), has been validatedThe protocol for this prospective, multicentre observational cohort study has been published previously.Between May 2015 and January 2017 we enrolled consecutive patients attending participating centres in both primary and secondary care, for whom ABPM was considered appropriate. Eligible participants were those undergoing ABPM as a result of routine blood pressure screening or monitoring in primary care or through referral to secondary care with suspected hypertension, newly diagnosed or treated hypertension, resistant hypertension, secondary hypertension, or other conditions requiring specialist advice. Anonymised data were collected on all patients fulfilling the eligibility criteria. We excluded patients if they lacked basic clinical information, did not have multiple clinic blood pressure readings recorded (obtained on at least three occasions within the same visit), and did not wear the ambulatory blood pressure monitor as instructed.All participants underwent ABPM, clinic blood pressure measurement, and collection of patient characteristics. The protocol provides information on the data collected for each participant,To capture as close to routine blood pressure measurements as possible, staff at participating sites were asked to measure clinic and ambulatory blood pressure, but we did not suggest or recommend a specific protocol for measurement. We defined routine blood pressure as readings taken by the consulting healthcare professional as part of standard clinical practice.For participants to be included in the study, we required a minimum of three clinic readings taken at the time of referral for ABPM or at fitting of the blood pressure monitor. Even though three measurements are recommended in guidelines,The triaging strategy applied an algorithm to three blood pressure readings taken at the clinic appointment, combined with information from an individual’s electronic health record: age, sex, body mass index, hypertensive and treatment history, and the presence of cardiovascular disease .The primary outcome was the proportion of participants whose hypertensive status was correctly classified using the triaging strategy compared with the reference standard of daytime ABPM (using a threshold for hypertension of ≥135/85 mm Hg).v secondary care), age (<65 years v ≥65 years), sex, smoking status (never or former smoker v current smoker), body mass index (<30 kg/m2v ≥30 kg/m2), and history of hypertension, diabetes, chronic kidney disease, and cardiovascular disease.We estimated the sensitivity (for detecting hypertension in those with the condition), specificity (for ruling out hypertension in those without the condition), and positive and negative predictor values, and we compared these with guideline strategies for measuring blood pressure from the United Kingdom,We used descriptive statistics to describe the number of patients classified with sustained hypertension, white coat hypertension, normotension, and masked hypertension with the triaging approach (the primary outcome) compared with daytime ABPM as the reference standard. These were used to calculate the sensitivity, specificity, and positive and negative predictor values of the triaging approach, and the total proportion of participants with correctly classified hypertensive status and proportion that would have been referred for ABPM.To examine model performance, we constructed a logistic regression model with true hypertension (defined by daytime ABPM) as the dependant outcome variable and classification using the triaging approach as the independent predictor variable. From this model we estimated the area under the receiver operating characteristic (AUROC) curve statistic. Further analyses were conducted examining the primary outcomes using different definitions of clinic and ambulatory blood pressure (see online appendix).Post hoc analyses were undertaken to examine performance of the PROOF-BP algorithm on its own  and this was compared with other blood pressure measurement strategies (employed without ABPM).All analyses were conducted using STATA version 13.1 . Results are presented as means or proportions, with standard deviations or 95% confidence intervals, unless stated otherwise.Based on the original validation of the PROOF-BP prediction model,Patients with a history of hypertension were approached to discuss the study at the design phase of the project. In particular their opinions were sought on the methods of recruitment and patient facing study literature, before ethical and NHS R&D applications.Ten general practice surgeries and one hospital trust participated in the study. A total of 897 patients attended participating sites for clinic blood pressure measurement and ABPM during the study period. In total, 10 patients (1.1%) were ineligible: eight had missing blood pressure readings, one was aged less than 18 years, and one had missing clinical information . The remMean age was 52.8 (SD 16.2) years, 53.8% were women, and 14.0% were current smokers . Mean clThe triaging strategy  predicted true blood pressure  with a low error rate  . The triThe triaging strategy had a sensitivity of 97%  and specificity of 76% , and an AUROC of 0.86  for predicting true hypertension . It woulv 42% secondary care) and in those patients with no history of hypertension (62% v 44% history of hypertension) or prescription for antihypertensives (56% v 44% existing prescription) (The sensitivity of the triaging strategy was consistently high (95-100%) regardless of the setting, underlying prevalence of hypertension, subgroup population, or clinic blood pressure monitor used , fig 2. ription) .v. 87.5% night time readings), reflecting the different diagnostic thresholds used to define night time hypertension. The performance of the PROOF-BP algorithm was reduced when utilised without ABPM—that is, not as a triaging tool (eTable 8).The triaging approach performed consistently well in terms of hypertensive classification and lower utilisation of ABPM, regardless of the number of clinic blood pressure readings or period of 24 hours used to define ambulatory blood pressure (eTable 7). The approach did result in fewer patients being correctly classified according to night time blood pressure  for suspected or poor control of hypertension in real world settings. In a population being referred for ABPM, use of a triaging strategy captured nearly all patients with hypertension while resulting in half the number of referrals for resource intensive ABPM compared with usual care. However, such a strategy would misclassify as hypertensive, one in four of those without hypertension, potentially leading to unnecessary treatment. Used in a diagnostic situation in people without a previous diagnosis of hypertension, this misclassification of normotensive people was reduced to one in seven. These findings suggest the potential for diagnosing and managing hypertension using a triaging strategy to reduce the need for ABPM.This study was conducted according to a prepublished, peer reviewed protocol, with analyses undertaken in line with a prespecified analysis plan. The study included 98.8% of patients attending routine practice during the study period, suggesting it is likely to be representative of the target population (those who have an indication for ABPM) and was sufficiently powered to examine key outcomes.Unlike the previous studies in which the PROOF-BP triaging algorithm has been previously validated,The study included patients in whom the treating doctor felt ABPM was appropriate. In most cases this was owing to suspected hypertension or apparently uncontrolled blood pressure (eTable 4). This included patients with no history of hypertension  and those already prescribed blood pressure lowering drugs . The findings are therefore widely applicable to patients undergoing diagnosis and also applicable to those in whom the consulting doctor is considering treatment intensification but is unsure if this is appropriate. Although additional monitoring is not recommended in those with normal blood pressure,Few studies have previously identified an effective method for triaging patients for out-of-office blood pressure monitoring, and none have externally validated such a method using data from a routine clinical setting. The original PROOF-BP derivation paper reported good performance of the algorithm when used in combination with ABPM, with a sensitivity of 96% and specificity of 87%. One study examined the optimal threshold for referral for out-of-office monitoring based on automated clinic blood pressure levels in patients with normal clinic pressure for detection of masked hypertension.Use of a triaging approach would substantially reduce the proportion of people requiring ABPM at the expense of “over treating” a small proportion of individuals. The triaging algorithm performed consistently across sensitivity analyses and subgroup analyses. Because the triaging mechanism is based on thresholds of adjusted clinic blood pressure, scenarios where a greater number of individuals have blood pressures close to the diagnostic threshold for hypertension are likely to result in greater numbers of patients being referred for ABPM. The greater the use of ABPM, the more accurate the triaging strategy was . Where ABPM facilities are widely available, clinicians may therefore want to consider additional use of ABPM, where the risk of misdiagnosis is great but an individual is not within the triaging range. This could include younger, lower risk people for whom a lifetime of treatment may have a larger impact on their quality of life. However, in low and middle income countries where resources might be more stretched, the PROOF-BP triaging approach represents an alternative option that maximises the benefits of ABPM and targets those with the most to gain.https://sentry.phc.ox.ac.uk/proof-bp) and could be incorporated into clinic computer systems, smartphone blood pressure management apps, and Bluetooth enabled monitors, providing general practitioners and hospital doctors with instant feedback and management recommendations in terms of referral or treatment.These data suggest that the PROOF-BP triaging strategy may be used in both primary and secondary care wherever ABPM is being considered to rule out white coat hypertension. To facilitate uptake, the algorithm is freely available as a calculator online  is the recognised standard for measuring blood pressure and diagnosing hypertensionABPM is currently recommended for all patients with raised screening clinic pressure, but triaging patients for this additional monitoring may be a more effective strategyBlood pressure readings taken in research studies are not always comparable to those taken in clinical practice where there may be suboptimal measurement technique and rounding biasThe PRedicting Out-of-OFfice Blood Pressure (PROOF-BP) algorithm accurately classified hypertensive status with half the utilisation of ABPM compared with usual careThe triaging strategy resulted in 435 participants  being referred for ABPM and the remainder managed on the basis of their clinic measurements; 69/435  would have received treatment deemed unnecessary had they received ABPMThis strategy can be used to triage for ABPM in both a primary and secondary care and for diagnosis or management of hypertension


To investigate the effect of mitochondrial dysfunction on the autoregulation of blood flow, by measuring levels of glutathione, an indicator of mitochondrial dysfunction, in glaucoma patients.Fifty-six OAG patients and 21 age-matched controls underwent a blood assay. Mitochondrial function was measured according to the levels of total glutathione (t-GSH), reduced GSH (GSH), and oxidized GSH  in peripheral blood mononuclear cells. Ocular blood flow in the optic nerve head was assessed with laser speckle flowgraphy parameters, including acceleration time index (ATI). We determined correlations between these measurements and other clinical parameters. Furthermore, we investigated the association between glutathione levels and glaucoma with a logistic regression analysis. Finally, we calculated the area under the receiver operating characteristic (ROC) curve in order to determine the power of redox index (the log GSH/GSSG ratio) to distinguish the groups.OAG patients demonstrated significantly higher GSSG levels and a lower redox index than the controls , but total GSH and reduced GSH levels were similar in the OAG subjects and controls . Additionally, redox index was significantly correlated with mean deviation (MD) of the visual field  and ATI . Multiple linear regression analysis showed that redox index contributed to MD (p = 0.02) and ATI (p = 0.04). The receiver operating characteristic curve (AUC) analysis suggested that redox index could differentiate between control eyes and eyes with glaucoma . The cutoff point for redox index to maximize its sensitivity and specificity was 2.0 .These results suggest that redox index is lower in OAG patients than in controls. Thus, it is possible that mitochondrial dysfunction contributes to glaucoma pathogenesis by causing vascular alterations. Glaucoma is the second most common cause of blindness worldwide; it is characterized by retinal ganglion cell (RGC) death and irreversible visual loss. Aging anGlutathione (GSH), a tripeptide consisting of glycine, cysteine and glutamic acid, prevents the effect of ROS when it changes its formation from reduced GSH to oxidized GSH . NormallyFifty-six OAG patients and 21 age-matched controls were recruited from Tohoku University Hospital, Miyagi, Japan, between December 2016 and February 2017. The procedures in this study followed the tenets of the Declaration of Helsinki and were approved by the institutional review board of Tohoku University Graduate School of Medicine. Written informed consent was obtained from all participants at the time of blood sample collection. Control subjects were recruited from patients who visited Tohoku University Hospital for treatment of diseases other than glaucoma, such as cataract, epiretinal membrane (ERM), dry eye, and traumatic optic neuropathy. No control subject had any history of elevated IOP or any apparent glaucoma. We selected the healthy contralateral eye of each control subject for inclusion. If both eyes had cataract or ERM, we chose the eye with better visual acuity. This study’s ocular exclusion criteria included narrow iridocorneal angles, evidence of secondary open-angle glaucoma, and other non-glaucomatous ocular diseases, including diabetic retinopathy, proliferative vitreoretinopathy, and age-related macular degeneration. We also excluded patients with a history of any chronic systemic disease with presumed abnormal circulating glutathione levels, including cancer, neurodegAll subjects underwent a baseline examination, including best-corrected visual acuity measurements , measurement of axial length (AL) with the Zeiss IOL Master , slit lamp examination, funduscopic examination, gonioscopy, IOP measurement, optical coherence tomography , visual field testing and measurement of systemic variables. IOP was measured with Goldmann applanation tonometry. Circumpapillary retinal nerve fiber layer thickness (cpRNFLT) was measured with spectral-domain OCT. The visual field was analyzed with the 24–2 Swedish interactive threshold algorithm standard program of the Humphrey Field Analyzer . The systemic blood pressure (BP), and heart rate (HR) were recorded in the left brachial artery at the height of the heart with an automated BP monitor  with the subject in a sitting position. Mean arterial blood pressure (MAP) and ocular perfusion pressure (OPP) were calculated as follows: MAP = diastolic BP + 1/3 (systolic BP–diastolic BP), and OPP = 2/3 MAP–IOP.The principles of laser speckle flowgraphy  have been described in detail previously. In brief2+. In order to avoid variations and GSH loss, all equipment and samples were maintained at 4° C in ice, including the GSH assay, and pH was maintained in the acidic range. End End25] E-). When this peroxynitrite reaches the RGCs, it can cause apoptosis if superoxide radicals (O2-) have been generated by reperfusion injury.[Vascular dysregulation, as described above, not only damages RGCs but also activates glial cells that support the RGCs, such as astrocytes., 52 Actin injury. Additionn injury. This seqn injury. This mecAlthough our method was quantitative and we consider that it was reasonable, there were several limitations. First, although we never collected blood from glaucoma patients sooner than 3 hours after the last meal, we did not completely standardize the condition of the subjects before blood sample collection, and factors such as diet, time when the blood test was performed, and lifestyle might have influenced the GSH concentration in the samples. Second, In conclusion, our study demonstrated that mitochondrial function was lower in OAG patients than in controls. Furthermore, our findings suggest that mitochondrial dysfunction might contribute to glaucoma pathogenesis by causing vascular alterations. Increasing evidence shows that mitochondrial dysfunction plays a key role in the etiology of glaucoma: therapies targeting the mitochondria are therefore a promising approach to develop new treatments for glaucoma.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Pheochromocytomas and paragangliomas (PPGLs) are rare catecholamine-secreting neuroendocrine tumors of the adrenal medulla and sympathetic/parasympathetic ganglion cells, respectively. Excessive release of catecholamines leads to episodic symptoms and signs of PPGL, which include hypertension, headache, palpitations, and diaphoresis. Intraoperatively, large amounts of catecholamines are released into the bloodstream through handling and manipulation of the tumor(s). In contrast, there could also be an abrupt decline in catecholamine levels after tumor resection. Because of such binary manifestations of PPGL, patients may develop perplexing and substantially devastating cardiovascular complications during the perioperative period. These complications include hypertension, hypotension, arrhythmias, myocardial infarction, heart failure, and cerebrovascular accident. Other complications seen in the postoperative period include fever, hypoglycemia, cortisol deficiency, urinary retention, etc. In the interest of safe patient care, such emergencies require precise diagnosis and treatment. Surgeons, anesthesiologists, and intensivists must be aware of the clinical manifestations and complications associated with a sudden increase or decrease in catecholamine levels and should work closely together to be able to provide appropriate management to minimize morbidity and mortality associated with PPGLs. Pheochromocytomas (PHEOs) are rare catecholamine-secreting neuroendocrine tumors derived from chromaffin cells of the adrenal medulla 80–85%). Paragangliomas (PGLs) are extra-adrenal tumors, originating from similar cells present in both sympathetic and parasympathetic ganglion cells 15–20%) [5–20% [1]%. ParagaSince the pioneering work by Gagner et al. of the first laparoscopic resection of PHEO in 1992, surgical management of PHEO has considerably improved owing to the advancement in pre, intra, and postoperative care of these patients . PostopeTo our best knowledge, most of the published articles focus primarily on preoperative and intraoperative care of PPGL patients, whereas studies detailing postoperative management are only available from individual case reports. It is extremely important for physicians of PPGL patients to provide not only appropriate preoperative evaluation and treatment but also adequate postoperative care.In this article, we describe the medical approaches to treat these patients after tumor resection based on our unique, long-standing experience with these patients at the National Institutes of Health. Additionally, we present the notable complications physicians should become aware of, including those emergencies that require immediate attention by a well-trained and experienced endocrinologist working alongside intensivists. Finally, this article provides clinical caveats to practicing clinicians regarding postoperative management of these patients.1, α2, β1, β2) are the final target site of action for these catecholamines. Therefore, it is essential to recognize the impact of catecholamines from PPGL on specific organs  2017, hypertension is defined as blood pressure > 130/80 mmHg . Risk faOne possible mechanism behind hypertensive crisis during PPGL resection includes massive catecholamine release secondary to tumor manipulation. Watchful and precise resection, appropriate vasodilator use, and clear communication between the surgeons and anesthesiologists are helpful in minimizing intraoperative hemodynamic fluctuation ,23,24. O1-adrenoceptor antagonists such as doxazosin, prazosin, and terazosin. These are predominantly used to manage perioperative hypertension following PPGL resection [2-adrenoceptor blockade. Therefore, it is recommended to be used in combination with esmolol (Brevibloc), a rapidly acting cardio-selective β1-adrenoceptor antagonist [2-adrenoceptors located in the musculature of bronchi and blood vessels. Moreover, as patients’ oral intake improves, cardio-selective β1-adrenoceptor blockers such as metoprolol (Lopressor) 25–50 mg three to four times a day or atenolol (Tenormin) 12.5–25 mg once a day  orally are also administered to control catecholamines or α-adrenoceptor blocker induced tachyarrhythmia [α-adrenoceptor antagonists are categorized according to specific receptor activity: Non-selective antagonists such as phenoxybenzamine and phentolamine and selective αesection ,28. Howeesection . The mostagonist . The negtagonist . It is gtagonist ,32. At hrhythmia ,34.1- and non-selective β-adrenoceptor blocker with α to β blocking ratio of 1:4–7 [Labetalol (Normodyne or Trandate) is a combined selective αof 1:4–7 . Prolongof 1:4–7 . Poopalaof 1:4–7 . Labetalof 1:4–7 ,39.Although calcium channel blockers (CCBs) are less effective than α- and β-adrenoceptor blockers, it is an important class of drug used to manage hypertension following PPGL resection. They minimize complications related to catecholamines overload, such as coronary vasospasm . CCBs inNitroglycerin is an antianginal drug and a more potent venous dilator than arterial dilator. However, arterial dilation occurs at higher doses . It has Hydralazine (Apresoline) use is very limited in the management of postoperative hypertension following PPGL resection due to its long duration of action resulting in hypotension and reflex tachycardia . HydralaMagnesium (Sulfamag) is considered safe and potent supplemental medication to block catecholamine action, bringing hemodynamic control in adults and children undergoing PPGL resection . MultiplPersistent hypertension following PPGL resection might be related to coexistence of essential hypertension. Oral antihypertensive medications, preferably a preoperative antihypertensive medication regimen, should be used under close monitoring to make any necessary adjustments in dosage. After discharge, a patient is followed by his or her primary care provider. Approximately six weeks later, laboratory evaluation is performed to evaluate for the presence of catecholamines and metanephrines in blood and urine, thus, identifying the presence of residual tumor, tumor at an unknown location, or metastatic tumor. However, if laboratory results are negative, a diagnosis of underlying essential hypertension is established, and antihypertensive medications are given as necessary.1-adrenoceptor mediated vasoconstriction [1-adrenoceptor blockers.Hypotension is defined as blood pressure below 90/60 mmHg or any degree of low blood pressure leading to organ hypoperfusion or end-organ damage. Potential risk factors attributed to postoperative hypotension following PPGL resection include chronically low circulating plasma volume, an abrupt decrease in serum catecholamine levels, down-regulation of adrenoceptors, increased blood loss, and cardiogenic or septic shock . Indepentriction . Moreovetriction ,62. NeveFirst line management for postoperative hypotension following PPGL resection includes vigorous intravenous fluid administration . If fluiFluid loss during the postoperative period results from: (1) Surgical site bleeding or occult bleeding, (2) suction drainage , (3) third spacing of fluid, i.e., capillary leak and extravasation of protein-rich serum into the interstitial spaces of the soft tissues, organs, deep space cavities , or retroperitoneum, (4) insensible or evaporative losses, and (5) loss of systemic vascular resistance after sudden withdrawal of catecholamines following tumor resection . First l1-adrenoceptor and mild to moderate β1-adrenoceptor and minimal β2-adrenoceptor agonist effects [1-adrenoceptor mediated augmentation of venous return [NE (Levophed) is an α- and β-adrenoceptor agonist with stronger affinity towards α effects . It is c effects . As the s return . NE infu1-adrenoceptor effect) and cardiac output (β1-adrenoceptor effect) [EPI  is an α- and β-adrenoceptor agonist with dose-dependent actions. Mixed actions of epinephrine cause an increase in both mean arterial blood pressure by vasoconstriction (α effect) ,69. The  effect) . It is c effect) ,72. Adve1), and V2 receptors in the following ways: (1) Increased systemic vascular resistance by V1 receptor stimulation on arterial smooth muscle cells and (2) water reabsorption at collecting ducts by stimulation of V2 receptors in kidney. Additionally, vasopressin may increase responsiveness of catecholamines allowing lower doses of adrenergic drugs to be used [Vasopressin , a nonapeptide hormone, is released from the posterior pituitary gland in response to increased plasma osmolality, decreased intravascular volume and low blood pressure. Vasopressin restores water balance and blood pressure by acting on vasopressin 1  and bradyarrhythmias (<60 beats/min). Tachyarrhythmia is commonly documented following PPGL resection from either increased catecholamine levels or inotropes used to correct postoperative hypotension . Other iResidual anesthetic effects and opioids and analgesics used in the postoperative period makes it difficult to identify the signs and symptoms of unstable tachyarrhythmia. Therefore, a multidisciplinary team of physicians, including intensivists, surgeons, anesthetists, cardiologists, and endocrinologists are required to manage these patients.In stable patients with narrow, regular (<0.12 sec) QRS complex that is not sinus tachycardia (non-sinus tachycardia), vagal maneuvers such as Valsalva or carotid sinus massage can be executed. If rhythm is regular, adenosine 6 mg rapid intravenous push is injected followed by a bolus dose of 12 mg when no response is observed within one to two minutes. An additional dose of 12 mg is given in resistant cases. Usually, adenosine is helpful to effectively terminate and/or diagnose tachyarrhythmia. If tachycardia resumes or is not responsive to adenosine, treatment with longer-acting atrioventricular (AV) nodal blocking agents such as diltiazem or β-adrenoceptor blocker is required. Stable patients with irregular narrow complex tachycardia usually have either atrial fibrillation or atrial flutter requiring an expert consultation and use of rate-controlling agents such as diltiazem, β-adrenoceptor blockers or antiarrhythmics such as amiodarone. Diltiazem (Cardizem) is given intravenously at a starting dose of 0.25 mg/kg over two minutes followed by a maintenance dose with an infusion at 5–15 mg/h. Commonly used β-adrenoceptor blockers to control tachyarrhythmia include metoprolol  and esmolol . Esmolol (Brevibloc) is loaded initially as 500 µg/kg over one minute with maintenance doses of 50–200 µg/kg/min and repeat bolus doses between each dose increase. However, a major drawback of using esmolol is that arrhythmias may recur after discontinuation. Therefore, longer acting β-adrenoceptor blocker, metoprolol tartrate (Lopressor), is used and given at a bolus dose of 2.5–5.0 mg intravenously over two minutes with repeated doses every 10 min up to a maximum three times before using longer intervals (every six to eight hours) . These mFor hemodynamically stable patients with wide complex tachycardia, 12-lead electrocardiogram (EKG) and expert consultation are obtained. Amiodarone is often the first medication used if there are no contraindications  are observed . RecommeFor prolonged, refractory or hemodynamically compromising tachyarrhythmias, electrical cardioversion is considered in combination with antiarrhythmics. If a patient has pulseless electrical activity , immediaf current in the sinus node, resulting in decreased heart rate without compromising myocardial contractility and cardiovascular hemodynamic status [It is not unusual for sinus tachycardia to remain uncontrolled despite using all drugs including esmolol, metoprolol, and diltiazem. Therefore, another therapeutic approach may be considered. Recently, ivabradine has been introduced to treat patients with heart failure and sinus tachycardia who failed or were intolerant to β-adrenoceptor blockers. Ivabradine selectively inhibits Ic status . Thus, Ic status ,81.In contrast, bradyarrhythmia’s after PPGL resection might result from sinus node or atrioventricular node dysfunction caused by progression of native conduction disease, overdose of rate controlling agents, i.e., β-adrenoceptor blocker, and metabolic or electrolyte disturbances. Bradycardia is defined in postoperative PPGL patients as heart rate below 60 beats/min. Therapeutic intervention is unnecessary for cases of transient bradycardia with no hemodynamic instability . DetrimeUnusually, bradyarrhythmias are unresponsive to atropine, DA, and EPI. Henceforth, transcutaneous or transvenous pacing becomes necessary.It is not uncommon to recognize myocardial injury following PPGLs resection. The mechanism behind injury to myocytes by increased catecholamines includes hemodynamic compromise (increased afterload), tachycardia, increased oxygen consumption, and coronary arterial vasoconstriction . MoreoveHeart failure may also be seen as one of the rare complications seen following PPGL resection. Myocardium may have been dependent on the excess catecholamines prior to resection due to desensitized adrenoceptors, possible catecholamine-induced cardiomyopathy or non-ischemic cardiomyopathy. Reduced circulatory volume may also play a significant role in the development of heart failure following tumor resection . TherefoClinical course of patients with PPGL can be complicated by ischemic or hemorrhagic stroke. The main mechanisms include: (1) Thrombotic or embolic occlusion of cerebral artery secondary to increased platelet aggregation , (2) cerPatients with PPGL have a higher rate of ischemic stroke in comparison to hemorrhagic events in the postoperative period ,98. The Symptoms may range from headaches to motor and/or sensory deficits, to confusion and seizures. Recognition of these symptoms is challenging due to the concomitant use of anesthetic or sedative therapy in the postoperative period . The modFor patients that develop ischemic or hemorrhagic stroke following PPGL resection, it is advisable to provide a multidisciplinary patient-centered treatment plan including neurology, critical care, surgery, interventional neuroradiology, and anesthesiology.Surgical approaches for PHEO resection can lead to primary adrenal insufficiency (PAI) in order of ascending frequency: Cortical sparing vs. unilateral vs. bilateral adrenalectomy. Glucocorticoids supplementation is not routinely prescribed for patients undergoing unilateral adrenalectomy because the contralateral adrenal gland can maintain eucortisolemia . An indiWhen adrenal insufficiency is clinically suspected among patients undergoing bilateral adrenalectomy or in cases of subclinical or overt cortisol co-secretion, hydrocortisone 50 mg intravenous bolus is administered before anesthesia induction followed by 25–50 mg intravenously every eight hours thereafter, with tapering over the next 24–48 h. If no immediate postoperative complications develop, and patient can tolerate oral intake, transition to physiological oral maintenance dose with hydrocortisone 10–12 mg x body surface area (BSA) is recommended ,105. TheClinical surveillance for symptoms suggestive of AI during the postoperative period is paramount. In addition, morning serum cortisol measurements with adrenocorticotropic hormone (ACTH) levels and Cosyntropin stimulation test are routinely used to confirm the diagnosis of AI . CosyntrMineralocorticoid replacement therapy is indicated among patients with evidence of primary AI or after bilateral adrenalectomy. In these cases, fludrocortisone should be initiated when hydrocortisone dosages fall below 50 mg/day. Fludrocortisone is usually administered at dosages of 50–100 µg/day. Mineralocorticoid replacement is monitored based on the development of clinical symptoms like salt cravings, volume depletion, and orthostatic hypotension, followed by measurement of renin activity levels to a target goal in the upper end of the reference range without development of side effects .Preceding discharge of patients with a confirmed diagnosis of AI, education should be provided for early recognition of adrenal crisis and sick day rules. Patients should be equipped with a steroid emergency card to be placed in wallets, set up on smartphones, and a medical alert bracelet .Renal injury is a rare complication associated with PPGL resection. The mechanism of renal injury is due to massive catecholamine release in the postoperative period that can potentially lead to: (1) Stimulatory effects on renin activity and (2) hypertensive crisis from severe vasoconstriction leading to hypoperfusion at the renal bed, ischemia, and necrosis of skeletal muscles provoking rhabdomyolysis ,111. In Occasionally, “mass effect” from tumor may lead to renal ischemia causing direct compression of the renal artery or vasospasm secondary to catecholamines excess ,113,114.In scenarios of hypertension leading to acute kidney injury, antihypertensive therapy must be initiated as mentioned earlier in this review. In cases of severe rhabdomyolysis-related acute kidney injury, hemodialysis is recommended. Intravenous fluids must be used judiciously. Colloids, such as albumin 4%, are recommended in patients at risk or with pre-existent renal failure and low albumin levels. Nephrotoxic agents should be discontinued, and supportive care should be provided under nephrology guidance.PPGL patients can have glucose homeostasis abnormalities mediated by elevated catecholamine secretion leading to increased liver glycogenolysis, inhibited insulin secretion from pancreatic β-cells, and increased insulin resistance in the skeletal muscle. These pathological changes can lead to preoperative hyperglycemia . Sudden Classic symptoms of hypoglycemia  may be masked in the postoperative period due to residual effects of anesthesia, opioid, or β-adrenoceptor blocker use. If untreated, hypoglycemia may result in neuronal cell death and brain damage . ConsequTreatment considerations include administration of glucose tablets, glucose gels, or carbohydrate containing juices to provide 15–50 g of glucose. Moreover, if the patient is unable to tolerate oral intake, treatment with Dextrose 5% infusion should be started and titrated to a glucose goal of >100 mg/dL. In emergent situations, where treatment with oral or intravenous dextrose is not feasible, administration of 1 mg intramuscular glucagon should be considered ,124,125.1- and α2-adrenoceptor antagonist) at a dose of 1–5 mg intravenously is justified. Additionally, phentolamine is also helpful in controlling elevated blood pressure following PPGL resection [Increased catecholamine levels in the postoperative period following PPGL resection affects gastrointestinal smooth muscle cells and inhibits acetylcholine release from the parasympathetic nervous system, resulting in complications ranging from transient intestinal motility abnormalities to constipation, pseudo-obstruction, bowel infarction and perforation ,131. Moresection ,136. Nevesection ,139,140 Complications observed after any surgical procedure which were not mentioned earlier include nausea, vomiting, urinary retention, hemorrhage, and wound infection. These complications are not elaborately described here as they can be managed similarly to any other surgical procedures. Meticulous monitoring along with the skills of a multidisciplinary team of physicians and appropriate nursing care, together with patient cooperation are helpful for faster recovery with no or minimal complications in the postoperative period.PPGL patients are at a significant risk of bleeding, which is difficult to be identified as hypotension. It is not uncommon after tumor resection. Elevated blood pressure as a result of higher catecholamines causes hemorrhage either intra or postoperatively. Precise surgical technique is crucial to avoid redundant blood loss intraoperatively. Therefore, an experienced surgeon is preferred to resect PPGL, minimize blood loss, and make use of meticulous surgical techniques to accurately scissor out tumor from a complex site. However, in the incidence of major hemorrhage, hemodynamic stability of the patient is assessed, and appropriate transfusion is given as per the needs of the patient and clinical judgment of the surgeon and anesthetist. Depending on the risk to benefit ratio, necessary medications would be stopped or continued during the perioperative period and if in doubt, a consult specialist opinion is considered.Surgical site/wound infection is a potential cause of morbidity and mortality in the postoperative period. Risk factors depend on location, nature of surgical wound/incision, and the procedure performed . PostopeUrinary retention, commonly regarded as a minor and trivial complication by surgeons, might cause increased restlessness, confusion, and delirium . A catecHemophilus influenzae, and Meningococcus [Splenectomy is required among those patients who are undergoing unilateral adrenalectomy on the left side due to the presence of a large-sized PHEO. Such patients need to be vaccinated preoperatively against pneumococcus, gococcus .Patients with PPGL resection must be managed appropriately in the intensive care setting during the postoperative period. Detailed physical examination and complete laboratory workup must be conducted at regular intervals to identify a patient at risk and provide treatment at the right point of time.


Chronic hepatitis C (CHC) is strongly associated with risks of cardiovascular diseases. The impact of direct acting antiviral (DAA) therapy on central blood pressure remains unclear. This investigation evaluates changes in central blood pressure following DAA therapy. One hundred and two DAA-treated patients were prospectively enrolled. Lipid profiles and pulse wave analysis of brachial artery by cuff sphygmomanometry including augmentation index (AIx), a parameter of central artery stiffness, were evaluated. All of the 102 patients achieved sustained virological response (SVR12). Cholesterol and LDL significantly increased following SVR12. Along with lipid changes, significantly higher central diastolic pressure  and AIx  were only observed in the advanced fibrosis patients. Co-morbid diseases, including hypertension , abnormal waist circumference , and metabolic syndrome  were associated with augmented AIx upon SVR12. The augmented central artery stiffness following viral eradication by DAA therapy may raise the concern of short-term cardiovascular risk in CHC patients. Additionally, long-standing infection increases the risk of extrahepatic events2. With respect to extrahepatic diseases of that are associated with HCV, a strong association exists between HCV infection and cardiovascular diseases, including coronary artery diseases3, carotid atherosclerosis4, and cerebral vascular diseases4.Hepatitis C virus (HCV) infection is a major health issue globally. Chronic infection leads to a risk of the development liver complications, including liver cirrhosis and hepatocellular carcinoma, and it increases liver-related morbidity and mortality5. PWA can be used to obtain central blood pressures, augmentation pressure, and the augmentation index (AIx), which are associated with risk of cardiovascular diseases7. Chronic hepatitis C (CHC) patients are known to exhibit an increased pulse wave velocity8. and our earlier investigation demonstrated that a higher arterial stiffness index and a lower compliance index were independently associated with CHC infection9. However, little is known about the temporal association of central blood pressures with CHC treatment. Therefore, the relationship between changes in PWA parameters and HCV eradication by direct acting antivirals (DAA) in CHC patients is both interesting and important.Pulse wave analysis (PWA) that measures by cuff sphygmomanometry of the brachial artery is used to evaluate changes of waveform morphology throughout the arterial system. The shape of pressure waveform changes from the aorta to distal arteries with constant diastolic pressure but a higher increased systolic pressure in the brachial artery than aorta. This feature is an augmentation of systolic pressure arises from an increase in arterial stiffness as the wave moves away from the aortaThe objectives of this investigation are to evaluate the changes in central blood pressure related parameters with HCV eradication by DAA therapy in CHC patients, and to examine the impact of advanced fibrosis on the parameters in PWA.2), dyslipidemia, and coronary artery disease did not differ between patients with non-advanced fibrosis and those with advanced fibrosis.Table Nighty patients 88.2%) had undetectable serum HCV RNA at week 4 of DAA therapy. All 102 patients (100%) achieved SVR12. Liver stiffness declined significantly from 15.4 ± 11.2 kPa at baseline to 11.8 ± 8.6 kPa at SVR12 (p < 0.001). At baseline, levels of insulin, HbA1c, HOMA-IR, HOMA-β, cholesterol, triglyceride, LDL, and HDL were similar across fibrosis stage . AIx was correlated with age  and was significantly higher in females than in males . PWA related parameters before and after HCV eradication were compared. Of note, all of these parameters tended to be higher at SVR12 than before DAA therapy. Only AIx were significantly increased  rather than in those with dyslipidemia . Other associated factors, including tobacco use, pre-existing chronic kidney disease, and pre-existing coronary artery diseases, were also analyzed. No significant changes of AIx were observed in patients with tobacco use , pre-existing chronic kidney disease , and pre-existing coronary artery disease  following viral clearance. Multivariate linear regression analysis was performed to identify metabolic factors associated with increased AIx. The only independent metabolic factor was absence of DM (p = 0.05). Other factors including dyslipidemia (p = 0.657), hypertension (p = 0.184), and abnormal waist circumference (p = 0.823) did not show significance.11. Our earlier study revealed that DAA treatment increased the loading capacity of triglyceride and cholesterol per very low-density lipoprotein of cholesterol particle, which transports lipid from liver to circulating blood12. The cholesterol loading capacity of LDL in the plasma also increased. The rapid changes in cholesterol and LDL levels were probably caused by increased delivery of lipid from liver to circulating blood following viral eradication by DAA therapy.DAA is a short-duration and highly efficient treatment for chronic hepatitis C; viral eradication is accompanied by lipid changes. In this investigation, the cholesterol and LDL levels of patients with non-advanced fibrosis and those with advanced fibrosis were significantly increased upon SVR12. These results are consistent with those in previous reports13. One recent study investigated the association of LDL with blood pressure and found that a high LDL level in non-statin users was associated with increased brachial and central blood pressures, with the greatest effect on central diastolic pressure14. LDL is known to affect detrimentally endothelial function, and elevated serum LDL levels result in higher vascular tone via upregulation of vascular Angiotensin II receptor gene expression by LDL in vascular smooth muscle cells15 and a reduction in the intracellular concentration of nitric oxide in endothelial cells16. These pathways may be consistent with findings of this investigation that increased LDL levels are associated mostly with an elevated steady component of central blood pressure, which is dominated by arterial resistance18. In this investigation, PWA of the brachial artery was used to obtain central (aortic) blood pressure and AIx. AIx is derived as augmentation pressure divided by central pulse pressure. An important finding of this investigation is the significant increase of AIx, a parameter of aortic stiffness, upon DAA therapy and the achievement of SVR12. Therefore, we speculate that the increased AIx in CHC patients who cleared HCV as a result of a short period of DAA therapy is partially, at least, mediated by increased LDL levels, which increased central diastolic pressure and reduced central pulse pressure. A mechanism that is not LDL-related may be involved. HCV-induced inflammation may have a role. In this investigation, the unchanged hsCRP upon SVR12 revealed a persistent inflammation reaction following HCV eradication. A cytokine and chemokine study showed that successful DAA therapy reduced the levels of up-regulated inflammation mediators, but not recovered to those of healthy controls19. Some mediators did not recover, or even remained elevated during or after therapy20. The complex presentations may predispose patients to persistent inflammation, even following HCV eradication. Along with increased LDL, persistent inflammation following HCV eradication may have a supplemental role in increased central diastolic pressure and aortic stiffness.LDL is strongly associated with a risk of cardiovascular diseases21, blunted response of central blood pressure to glucose22, and lower reflection wave magnitude as a result of a reduction of the impedance mismatch of the large artery and distal arteries23 may have contributed to the unchanged AIx in patients with DM following viral eradication. All of the DM patients were treated by oral hypoglycemic agents or insulin therapy. Controlling of sugar was not likely to be beneficial to aortic stiffness following SVR12 in this investigation. Similar to patients with DM, significantly higher AIx was observed only in patients with non-dyslipidemia patients. In this investigation, all patients with dyslipidemia took statin. For statin users, reduction of LDL level was associated with a fraction of the reduction of blood pressure and seemed to be associated mostly with the improvement in central steady (diastolic) pressure14, and then contributed to a lesser degree of changes in AIx following eradication of HCV. In this investigation, patients with hypertension all treated by anti-hypertensive drugs and also showed higher aortic stiffness than non-hypertension patients that indicated the possible detrimental impacts on diseased arteries of hypertensive patients. Other factors including tobacco use, pre-existing chronic kidney disease, and coronary artery disease did not show impact on AIx following viral clearance. However, the patient number was small that could not make solid conclusion in this investigation.A significant increase in aortic stiffness was observed only in patients with advanced fibrosis, and not in those with non-advanced fibrosis. In this investigation, patients with advanced liver fibrosis were older than those with non-advanced fibrosis. However, before DAA therapy, their AIx values did not differ . Cholesterol and LDL levels increased significantly, regardless of fibrosis stage. However, brachial and central diastolic pressures were significantly elevated only in advanced fibrosis patients, indicating that the detrimental effects of LDL on central blood pressure and aortic stiffness seemed to be greater in advanced fibrosis patients. Co-existing diseases or metabolic syndrome were also associated with higher AIx in advanced fibrosis patients. In advanced fibrosis patients, hypertension, abnormal waist circumference, and metabolic syndrome all increased aortic stiffness following viral eradication. However, AIx in patients with DM did not differ before therapy and upon SVR12. A lesser degree of impairment of left ventricular systolic function24. These effects may degrade cardiac function. Furthermore, an increase in aortic stiffness reduces aortic pressure during diastole reducing the coronary perfusion pressure and thereby predisposing patients to myocardial ischemia25. Patients with pre-existing heart failure or coronary artery disease may be at a risk of developing associated events during treatment and a short follow-up period. Experiences of serious adverse events during or after DAA therapy, especially cardiovascular events, which had previously not been recognized as being related to DAA therapy should be re-examined. Patients with simultaneous advanced fibrosis and disease background of cardiovascular or metabolic diseases that may predispose them to deterioration of cardiovascular function should be closely monitored throughout DAA treatment and a short post-therapy follow-up period.The long-term implications of the above findings remain to be elucidated. An increase in AIx, a parameter of aortic stiffness, theoretically causes an increase in the afterload of heart and boosts aortic and left ventricular pressure in systole by causing the early return of the reflection wave26, long-term findings concerning central arterial stiffness may differ from the short-term observations in this study and warrant further investigation. In this investigation, associated endothelial or cytokine factors following the course of DAA therapy were not evaluated. Further studies concerning these issues are needed. Finally, the number of patients with non-advanced fibrosis is limited, more patients enrolment is required to obtain more solid results.This study has limitations. The follow-up period was short. Lipid and arterial stiffness should be measured over a longer period after SVR12 is achieved. Based on the beneficial effects of interferon/ribavirin treatment on cardiovascular riskIn summary, the data herein suggest that LDL levels increase, along with aortic stiffness, shortly after the eradication of HCV by DAA treatment. These results seem to contradict current knowledge of the long-term beneficial effects of curing HCV on cardiovascular risks. Further large-scale and long-term prospective investigations are required to address this interesting and important issue.Patients who had CHC and underwent DAA therapy at the National Cheng Kung University Hospital from February 2017 to October 2017 were prospectively enrolled in this study. CHC was defined as positivity for anti-HCV antibody for more than six months with the presence of serum HCV RNA. Medical history, including co-morbid diseases, alcohol consumption, smoking status, co-medications, and CHC treatment history were reviewed. Eligible patients were followed up for 12 weeks following the completion of DAA therapy. Fasting blood sampling was carried out, and measurements of liver stiffness, waist circumference, body weight and body height made before therapy and at SVR12. Waist circumference ≥90 cm in males or ≥80 cm in females was regarded as abnormal. Patients who exhibited three of the five following characteristics were diagnosed as having metabolic syndrome; abnormal waist circumference, systolic blood pressure ≥130 mm Hg or diastolic blood pressure ≥85 mm Hg or currently taking blood pressure-lowering agents, high-density lipoprotein of cholesterol <40 mg/dL in males or <50 mg/dL in females, fasting blood sugar ≥100 mg/dL or currently taking diabetes medications, and triglyceride ≥150 mg/dL. In addition, Homeostasis Model Assessment (HOMA) of insulin resistance (IR) and beta cell function (β) were evaluated. The equation of HOMA-IR and HOMA-β was (fasting sugar × insulin)/405 and (360 × insulin)/(fasting sugar-63), respectively. Advanced liver fibrosis was defined as liver stiffness ≥9.5 kPa, measured by transient elastography  or Fibrosis-4 score ≥3.25. The Research Ethics Committees of the National Cheng Kung University Hospital approved this investigation (No: A-ER-105–479), which was conducted according to the guidelines of the International Conference on Harmonization for Good Clinical Practice. All patients provided written informed consent before enrollment.Blood samples were stored at −70 °C until testing. High-sensitivity C-reactive protein (hsCRP), lipid profiles including cholesterol, triglyceride, low-density lipoprotein of cholesterol (LDL), and high-density lipoprotein of cholesterol (HDL), and sugar profiles including fasting sugar, glycemic hemoglobin (HbA1c), and insulin were obtained both before therapy and 12 weeks after the end of treatment. Quantitation of HCV RNA was using the real-time polymerase chain reaction . Sustained virological response (SVR12) was defined as undetectable HCV RNA at 12 weeks after the end of DAA therapy.All tests were carried out in the morning (9:00~11:00am). Before any tests were performed, all subjects rested in a sitting position for five minutes in a quiet, temperature-controlled (26° ± 1 °C) room. PWA was carried out using a SphygmoCor XCEL PWA . A conventional brachial cuff on the right upper arm of each subject was used to measure brachial systolic and diastolic pressures, and a brachial waveform was thus captured. The brachial waveform was then analyzed using SphygmoCor to provide a central aortic waveform. Central blood pressure measurements, such as those of central aortic systolic blood pressure, central pulse pressure and AIx, were made. AIx measures the degree to which the peak of a measured pressure wave exceeds the peak of the incident pressure wave owing to the addition of the reflected pressure wave. The AIx depends on the timing and magnitude of the reflected waveform and is affected by the compliance and structure of vessels distal to the site of measurement. The SphygmoCor system uses the more common AIx as the augmentation pressure  divided by the pulse pressure , expressed as a percentage. The AIx ratio was controlled at a hear rate of 75 beats/min.The data were expressed as mean plus standard deviation. Continuous variables were compared by the Student t test and categorical variables were compared using the Chi-Square test or Fisher’s exact test. Comparisons of lipid profiles, sugar profiles, hsCRP, and parameters of arterial stiffness before treatment and 12 weeks following the end of treatment were carried out using the paired t test. The correlation between AIx and age was evaluated. Analysis of the associated metabolic factors of increased AIx was carried out by multivariate linear regression analysis. All tests were two-tailed. A p value of less than 0.05 was considered to indicate statistically significance.


Presepsin, a glycoprotein produced during bacterial phagocytosis, is used as a sepsis marker for bacterial infections. However, presepsin levels are affected by renal function, and the evaluation criteria according to kidney function or in chronic kidney diseases remain controversial. Furthermore, presepsin may be increased by sample stirring, but no studies have evaluated this effect.In this study, we excluded the effect of stirring by standardizing the blood collection conditions, analyzed the influence of kidney function on presepsin concentrations, and recalculated the reference range based on the findings. EDTA-whole blood from 47 healthy subjects and 85 patients with chronic kidney disease was collected to measure presepsin by PATHFAST. Presepsin was found to be significantly correlated with the levels of creatinine (r = 0.834), eGFRcreat (r = 0.837), cystatin-C (r = 0.845), and eGFRcys (r = 0.879). Furthermore, in patients with CKD, presepsin levels stratified by eGFRcys showed a significant increase in the CKD G2 patient group and with advancing glomerular filtration rate stage. The following values were obtained: Normal: 97.6 ± 27.4 pg/mL, CKD G1: 100.2 ± 27.6 pg/mL, CKD G2: 129.7 ± 40.7 pg/mL, CKD G3: 208.1 ± 70.2 pg/mL, CKD G4: 320.2 ± 170.1 pg/mL, CKD G5: 712.8 ± 336.3 pg/mL. The reference range, calculated by a nonparametric method using 67 cases of healthy volunteers and patients with chronic kidney disease G1, was found to be 59–153 pg/mL, which was notably lower than the standard reference range currently used. Presepsin concentrations were positively correlated with a few biomarkers of renal function, indicating the necessity to consider the effect of renal function in patients with renal impairment. Using the recalculated reference range considering kidney function may improve the accuracy of evaluating presepsin for diagnosis of sepsis compared to the standard reference currently in use. Recently, it was reported that granulocyte-mediated bacterial phagocytosis triggers elastase or cathepsin D to proteolytically cleave CD14 to produce presepsin and release it into the blood [Presepsin is a protein whose blood concentrations increase specifically during sepsis. Since its discovery in 2002 in Japan, presepsin has been widely used as a sepsis marker. Membrane-bound CD14, a surface antigen expressed on the cell membrane of monocyte macrophages and granulocytes, is a receptor for bacterial lipopolysaccharide (LPS), which activates cells via Toll-like receptor 4 [he blood . FurtherUnlike procalcitonin, which is conventionally used for sepsis diagnosis, presepsin responds very weakly to inflammation such as trauma and burn, and is considered highly specific for bacterial infection –7. CompaThe presepsin cut-off value for sepsis or infectious disease diagnosis is 400–700 pg/mL (500 pg/mL: Japan) . HoweverIn the current study, presepsin levels were measured in patients with CKD and analyzed for their relationship with the renal function index. Particularly, we focused on the relationship in early CKD stages. Additionally, by standardizing the blood collection conditions and excluding the effect of stirring, we established a reference range for evaluating the influence of kidney function.2 had abnormalities in the kidney structure such as albuminuria, urine sediment abnormalities, electrolytes, and other abnormalities due to tubular disorders or abnormalities detected by histology, structural abnormalities detected by imaging, or a history of kidney transplantation. Symptoms were present for >3 months.This study enrolled 85 outpatients with CKD who visited the Tokushima University Hospital from May 2017 to September 2017. All patients were over 18 years of age and had no history of dialysis or infection symptoms. Infection was diagnosed by the physician based on apparent clinical manifestations or laboratory results. We confirmed that patients with eGFR >60 mL/min/1.73 mSamples and data were also collected from 47 healthy volunteers without renal dysfunction as controls .Venous blood was collected after overnight fasting. Because the presepsin concentration is increased by strong agitation, samples after complete blood count measurement cannot be used. Thus, samples were collected into EDTA-2K blood collection tubes for presepsin measurement. At the time of blood sampling, five gentle inversions were performed . The bloThe estimated glomerular filtration rate (eGFR) of each participant was calculated using the equation provided by the Japanese Society of Nephrology as follows:2) = 194 × creatinine (mg/dL)-1.094 × Age-0.287 eGFRcreat (mL/min/1.73 m2) = eGFRcys  based on a chemiluminescent enzyme immunoassay with EDTA-whole blood.Recalculation of the reference range was performed using the nonparametric method. All values were listed in order according to the CLSI guidelines, and values of 2.5–97.5% were used as the reference range. Excision of extremes was not performed .This study protocol and consent procedure were approved by the Ethics Committee of Tokushima University Hospital (No. 2699) and performed in compliance with the Helsinki Declaration. Written informed consent was obtained from all patients.post hoc test for multiple comparisons. Correlation was evaluated by Spearman’s correlation coefficient by a rank test. Statistical analyses were performed with EZR  [P value < 0.05 was considered to indicate significance.All values are shown as the mean ± standard deviation. The results were analyzed as non-parametric variables using Mann-Whitney’s U test for comparison between two groups and using Kruskal-Wallis tests with Bonferroni , Japan) . A P valP = 0.01)  was significantly higher (410.1 ± 318.9 pg/mL) than that in the normal group (97.6 ± 27.4 pg/mL) ( = 0.01) .The presepsin levels were analyzed for their correlation with creatinine, eGFRcreat, cystatin-C, and eGFRcys. The correlation coefficients were as follows: creatinine, r = 0.834; eGFRcreat, r = 0.837; cystatin-C, r = 0.845; and eGFRcys, r = 0.879. All correlations were significant, and presepsin levels increased with deterioration of renal function .In patients with CKD, presepsin levels were stratified by GFR stage classified by eGFRcys . The preAs a result, presepsin levels showed a significant tendency to increase in the CKD G2 patient group, which is regarded as the mildly declining group, as the GFR stage increased. In contrast, in patients with CKD G1, in which renal function was maintained, there was no significant difference between presepsin levels in patients and control individuals.A total of 67 cases in the normal group and CKD G1 patient group were used to determine the reference range calculated using a nonparametric method. The recalculated reference range was 59–153 pg/mL, which was lower than the reference range of 98.3–314 pg/mL provided by the reagent manufacturer of PATHFAST.Presepsin levels are affected by altered renal function and sample agitation. However, in previous studies, the effects of agitation were not considered, and the possibility of a false high level could not be excluded.This study, which eliminated the influence of stirring and sampling, clarified that presepsin concentrations are strongly correlated with various renal function indices and tended to increase with renal function deterioration.The molecular weight of presepsin is approximately 13 kDa, which is similar to that of cystatin-C. Presepsin levels increase and accumulate in the blood when renal function is impaired in patients with renal disorders.As significant correlation was found for all indices examined in this study. Interestingly, eGFRcreat, which is generally used as a renal function index, rather than creatinine, showed the same correlation as creatinine. Because the presepsin level was not dependent on the individual’s sex or age, these factors were predicted to have no significant influence on the presepsin value. In the stratified comparison, a significant difference was found in both eGFRcreat and eGFRcys. However, for eGFRcys, the influence from a lower range was confirmed . This waAlthough presepsin showed an increasing trend in the early stage due to the deterioration of renal function, there was no significant difference between healthy subjects and patients in the CKD G1 group. Thus, the CKD G1 group may be diagnosed as normal. In patients in the CKD G2 group and higher stages, a significant increase in presepsin was observed even in uninfected cases. Sepsis diagnosis is not based on only one biomarker, but is a comprehensive diagnosis based on multiple markers and clinical symptoms. Presepsin levels must be carefully interpreted to diagnosis sepsis in patients with renal impairment.Although it has been reported that presepsin is related to age and hemoglobin levels, multivariate analysis using these factors as explanatory variables revealed no independent causality other than renal function . It is pAdditionally, false high values of presepsin arising due to vigorous agitation of the blood samples have been reported, preventing accurate clinical assessment. Mechanical stimuli such as agitation lead to the formation of macromolecular complexes, which reacts with the anti-presepsin antibody in the reagent in a larger molecular weight fraction. Although the false high value is considered to be due to cross-reaction with this polymer complex, the mechanism of complex formation has not been clarified. There are no efficient methods other than avoiding agitation of the sample at the time of blood collection/delivery to prevent these effects, necessitating extreme caution. Although doctors and nurses collect blood at the bedside with caution, fluctuations at the pre-measurement stage can be large, affecting the reliability of the results. Therefore, it is essential to educate clinical practitioners regarding specimen collection.In this study, recalculation of the reference range using data from the CKD G1 group and normal group indicated remarkably low values 59–153 pg/mL, which were half of the reference range currently in use. The sample group used to calculate the reference range by the reagent manufacturer did not consider the effects of renal function or agitation, and it is likely that these false high values were included. Therefore, this sample group may not be appropriate for calculating the reference range. In contrast, the sample group used for recalculation in this study excluded false high value cases caused by renal function and agitation, making it a more useful index.The new reference range obtained in this study can be used for the early diagnosis of sepsis in patients with normal kidney function. However, the new reference range obtained in this study did not meet the requirement for 120 cases recommended by CLSI guidelines and may not be sufficient evidence for establishing a new reference range . FurtherThis study had some limitations. First, the presence or absence of an infectious disease could not be completely excluded, as this factor was based on the judgment of the physician. Second, in the sample group examined in this study, there was an age difference between the normal group and CKD patient group. Although age was not an independent variable, normal groups with no difference in age should be considered as the control group.In conclusion, by excluding the effect of stirring during sampling, our study demonstrated that presepsin levels exhibit an increasing trend as GFR decreases. Currently, the false high values obtained using the standard reference range are largely ignored. Therefore, using the newly recalculated reference range, which excludes the effects of renal function and agitation, may improve the efficiency in sepsis diagnosis, particularly in the early stage. However, we recommend larger studies to confirm these findings and support alternate reference ranges.S1 FigP value was calculated using paired t test.(PDF)Click here for additional data file.S2 FigP value was calculated by Kruskal-Wallis one-way analysis of variance.P value adjustment was calculated using the Bonferroni method.(PDF)Click here for additional data file.S1 Table* Pearson`s correlation coefficients.(PDF)Click here for additional data file.