# Ã±ame

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## Anti-hypertensive and angiotensin-converting enzyme ...

Results: Our data showed that AME inhibited ACE activities in a dose-dependent manner, with an IC 50 of 1.85 ± 0.01 μg/ml. In comparison with mock, oral administration of AME reduced systolic blood pressure (SBP) levels in SHRs, and the level of SBP was decreased by 22.33 ± 3.61 mmHg at 200 mg/kg AME.the ACE inhibitory effect of AM and the presence of ACE inhibitory phytopeptide in AME supported the ethnomedical use of AM on hypertension..
Keyword: pmid:32119952, doi:10.1016/j.jep.2020.112724, Jing-Shan Wu, Jung-Miao Li, Tin-Yun Ho, Angiotensin-Converting Enzyme Inhibitors / pharmacology, Angiotensin-Converting Enzyme Inhibitors / therapeutic use*, Animals, Antihypertensive Agents / pharmacology, Antihypertensive Agents / therapeutic use*, Astragalus propinquus, Blood Pressure / drug effects, Drugs, Chinese Herbal / pharmacology, Drugs, Chinese Herbal / therapeutic use*, Hypertension / drug therapy*, Male, Mice, Inbred BALB C, Molecular Docking Simulation, Peptides / pharmacology, Peptides / therapeutic use*, Peptidyl-Dipeptidase A / metabolism, Rats, Inbred SHR, PubMed Abstract, NIH, NLM, NCBI, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, MEDLINE
From: pubmed.ncbi.nlm.nih.gov

Ethnopharmacological relevance: Hypertension is one of the common chronic health problems in the world. Astragalus membranaceus root (AM), also known as Huangqi, is a popular medicinal herb traditionally used to reinforce vital energy and modulate hypertension.

Aim of the study: This study was to reveal the anti-hypertensive activities and mechanisms of AM in spontaneously hypertensive rats (SHRs). Moreover, the presence of bioactive components in AM was further identified.

Materials and methods: We analyzed the effects of aqueous extract of AM (AME) on the regulation of blood pressure and angiotensin converting enzyme (ACE), the major target of anti-hypertensive drugs. Proteomic, bioinformatics, and docking analyses were performed to identify the anti-hypertensive bioactive peptides in AME.

Results: Our data showed that AME inhibited ACE activities in a dose-dependent manner, with an IC50 of 1.85 ± 0.01 μg/ml. In comparison with mock, oral administration of AME reduced systolic blood pressure (SBP) levels in SHRs, and the level of SBP was decreased by 22.33 ± 3.61 mmHg at 200 mg/kg AME. Proteomic analysis identified that an abundant 152-amino-acid putative protein kinase fragment accounted for approximately 11.7% of protein spots in AME. AM-1 (LVPPHA), a gastrointestinal enzyme-resistant peptide cleaved from putative protein kinase fragment, inhibited ACE activities, with an IC50 value of 414.88 ± 41.88 μM. Moreover, oral administration of AM-1 significantly decreased SBP levels by 42 ± 2.65 mmHg at 10 μmol/kg. Docking analysis further showed that AM-1 docked into the active site channel of ACE and interacted with Ala-354 in the active site pocket of ACE.

Conclusions: the ACE inhibitory effect of AM and the presence of ACE inhibitory phytopeptide in AME supported the ethnomedical use of AM on hypertension.

## Assessment of left ventricular myocardial work in Turner ...

Introduction. Turner syndrome (TS) is an X-chromosomal disease that occurs approximately in one out of 2,500 to 3,000 female newborns ().Girls and women with TS have an increased cardiovascular morbidity (): congenital heart defects such as the bicuspid aortic valve or the coarctation of the aorta are frequently seen among this cohort ().TS is also associated with …Assessment of left ventricular myocardial work in Turner syndrome patients: insights from the novel non-invasive pressure-strain loop analysis method.
From: qims.amegroups.com

Introduction

Turner syndrome (TS) is an X-chromosomal disease that occurs approximately in one out of 2,500 to 3,000 female newborns (1).

Girls and women with TS have an increased cardiovascular morbidity (2): congenital heart defects such as the bicuspid aortic valve or the coarctation of the aorta are frequently seen among this cohort (3). TS is also associated with multiple cardiovascular and cardiometabolic risk factors such as diabetes, lipid anomalies, arterial hypertension and excess weight (4). In addition, arterial stiffness, a predictive cardiovascular risk marker (5), has been shown to be elevated in TS patients (6-9).

The accumulation of the above-mentioned risk factors, which may also promote the arterial stiffening process of the small and major vessels, is assumed to increase left ventricular afterload and thus impair left ventricular function. Studies could demonstrate that TS patients display lower left ventricular function and higher left ventricular mass (10,11). However, in a recent study of our group, the previously described left ventricular dysfunction could not be confirmed through two-dimensional speckle tracking echocardiography (2DSTE) (6).

Recently, a new echocardiographic method has been introduced allowing the measurement of myocardial work and myocardial efficiency through left ventricular pressure-strain loops (12,13).

The aim of this study was to investigate whether alterations in myocardial work and work efficiency can be found in TS patients through left ventricular pressure-strain loop analysis (PSL) in comparison to conventional left ventricular global strain assessment.

Methods Ethical statement

This study was a retrospective analysis of prospectively collected data and was approved by the Ethics Committee of the Arztekammer des Saarlandes (State Chamber of Physicians of the German federal state of Saarland), Faktoreistrase 4, 66111 Saarbrucken, Germany, on March 23rd, 2018; approval statement No. 07/18. This study conformed to the standards set by the latest revision of the Declaration of Helsinki. Prior written informed consent was obtained from all patients or the parents or legal guardians of patients under legal age.

Study population

Thirty-eight TS patients and 19 healthy, age-matched controls participated in this study. Study subjects were recruited between November 2016 and April 2018. TS patients examined for clinical routine inspection at the departments of pediatric cardiology and pediatric endocrinology of Saarland University Hospital were chosen. In addition, external TS patients were included in this study through collaboration with the German Turner Syndrome Association (Turner-Syndrom-Vereinigung Deutschland e.V.). The control group was composed of healthy, age-matched volunteers and patients with accidental heart murmurs in whom cardiac pathologies were ruled out by echocardiography and electrocardiography. Body surface area (BSA, m2) was assessed according to Mosteller’s formula (14). In study subjects <18 years of age, weight classification was evaluated according to body mass index (BMI, kg/m2) percentiles established by Kromeyer-Hauschild et al. (15). In study participants ≥18 years of age, normal weight was defined as BMI <25 kg/m2, overweight as BMI ≥25 kg/m2 but <30 kg/m2 and obesity as BMI ≥30 kg/m2.

Echocardiography

A 2.5–3.5 MHz phased array transducer with a Vivid 9 ultrasound system (General Electrics Healthcare, Fairfield, CT, USA) was applied for the entire echocardiographic examination.

Left ventricular dimensions

Left ventricular dimensions were obtained through M-Mode echocardiography in parasternal long axis view at the tip of the mitral valve. Left ventricular borders were evaluated at end-diastole (QRS complex in ECG) and end-systole (end of T-wave in ECG). The variables listed below were assessed: interventricular septum thickness at end-diastole (IVSd, cm), interventricular septum thickness at end-systole (IVSs, cm), left ventricular internal dimension at end-diastole (LVIDd, cm), left ventricular internal dimension at end-systole (LVIDs, cm), left ventricular posterior wall thickness at end-diastole (LVPWd, cm), left ventricular posterior wall thickness at end-systole (LVPWs, cm), left ventricular end-diastolic volume (EDV, mL), left ventricular end-systolic volume (ESV, mL), stroke volume (SV, mL), ejection fraction (EF, %), fractional shortening (FS, %), left ventricular end-diastolic mass (LV Mass, g). “Z-scores of cardiac structures” were measured according to Pettersen et al. for IVSd, IVSs, LVIDd, LVIDs, LVPWd and LVPWs (16). EDV, ESV, SV and LV Mass were put in relation to BSA.

End-systolic wall stress

End-systolic wall stress (ESWS, ×103 dynes/cm2), which is considered to reflect left ventricular afterload, was calculated according to Wilson et al. using the following formula (17,18):

TS patients with history of coarctation of the aorta or prior cardiac surgery were excluded from ESWS analysis.

Left ventricular systolic deformation

Left ventricular systolic deformation was evaluated through 2DSTE. Detailed information on methodological procedure of 2DSTE has been described in a recent publication of our group (6). In short: A constant frame rate of 60–90 fps was applied during echocardiographic examination of the left ventricle in apical two chamber view, apical three chamber view and apical four chamber view. We then utilized a separated workstation (EchoPAC PC version 202, General Electrics Healthcare) to analyse recorded loops offline. The endocardium of the left ventricle was marked precisely, and the region of interest was adapted to the myocardial thickness. Time of end-systole was represented by aortic valve closure. The software then generated a GLPS for the apical two chamber view (GLPS_A2C, %), the apical three chamber view (GLPS_A3C, %) and the apical four chamber view (GLPS_A4C, %). In addition, an average (GLPS_AVG, %) of the three assessed chamber views (GLPS_A2C, GLPS_A3C, GLPS_A4C) was determined. TS patients with history of coarctation of the aorta or prior cardiac surgery were excluded from left ventricular 2DSTE analysis.

Left ventricular myocardial work and efficiency

To assess left ventricular myocardial work and efficiency, a non-invasive pressure-strain loop was generated (PSL). The procedure was executed offline through a semi-automatic software (EchoPAC PC version 202, General Electrics Healthcare). Brachial systolic blood pressure (SBP, mmHg) was measured simultaneously during echocardiographic examination. Left ventricular strain, which was required for PSL composition, was assessed through 2DSTE. Time of isovolumic and ejection phase was set manually by evaluating mitral and aortic valvular events through echocardiography in the apical three chamber view.

The left ventricular pressure curve generation was validated by Russell et al. through invasive cardiac catheterization. For the individual non-invasive PSL analysis the left ventricular pressure curve of reference is then adjusted by modifying its amplitude (SBP) and width (valvular events) (13).

The area within the PSL is considered as an index of global myocardial work (mmHg*%) (18). Global constructive work (mmHg*%) comprises myocardial work leading to myocardial shortening and thus to left ventricular ejection (18). Myocardial work that is not leading to left ventricular ejection is considered as global wasted work (mmHg*%) and includes dyssynchronous muscle fibres which are stretching during systole (13,18).

The ratio:   is defined as global myocardial work efficiency (%) (18). For better comparability of the TS and the control group, global myocardial work index, global constructive work and global wasted work were indexed to BSA (mmHg*%/m2). In addition, TS patients with history of coarctation of the aorta or prior cardiac surgery were excluded from PSL analysis. Figure 1 illustrates the non-invasive left ventricular PSL, myocardial work and myocardial efficiency.

Pulse wave velocity

Pulse wave velocity (PWV, m/s), a parameter to evaluate vascular stiffness, was evaluated non-invasively through an oscillometric blood pressure measuring device (Mobil-O-Graph, I.E.M. GmbH, Stolberg, Germany). Detailed information on methodological background has been described in a recent publication of our group (6). The Mobil-O-Graph has been validated through invasive catheter examinations (19). In addition, device specific PWV reference values exist for healthy pediatric and adult cohorts (20,21). Cuff sizes were chosen according to the left upper arm circumference. The PWV analysis was only executed after the study participant had been resting for at least 5 to 10 minutes in a seated position.

TS patients with history of coarctation of the aorta or prior cardiac surgery were excluded from PWV analysis.

Figure 2 represents schematically the influence of PWV on central hemodynamics.

Statistical analyses

For statistical analyses SPSS 24 (Released 2016. IBM SPSS Statistics for Windows, Version 24.0. IBM Corp., Armonk, NY, USA) was used. The Kolmogorov–Smirnov test was utilized to evaluate normality of continuous variables. Data are presented as mean ± SD or as median, minimum and maximum if not normally distributed. The independent-samples t-test was applied to compare continuous variables with normal distribution. Non-normally distributed continuous variables were compared using the Mann-Whitney U test. For correlation analyses, the Pearson correlation was utilized for normally distributed variables or the Spearman correlation for non-normally distributed variables. A P value <0.05 was considered as statistically significant.

Results Patient characteristics

The median age was 16.00 years for the TS group and 16.35 years for the control group. Both groups did not differ significantly in age. TS patients were, compared to controls, significantly smaller in height (P<0.001) and showed a significantly lower BSA (P=0.004). BMI (P=0.025) and resting heart rate (P<0.001) were significantly increased in the TS group. No significant differences were assessed in weight, SBP and DBP between TS patients and control subjects. Detailed information on patient characteristics are summarized in Table 1.

Karyotype and cardiovascular morbidity of TS patients

Out of 38 TS patients, 21 (55.3%) displayed a monosomy X, 12 (31.6%) a mosaic form of TS, and 2 (5.3%) structural chromosomal aberrations. Three (7.9%) TS patients had diagnosis of “TS unspecified (Q. 96.9)”.

Overall, 15 (39.5%) TS patients presented with congenital heart disease. Ten (26.3%) TS patients were diagnosed with bicuspid aortic valve and 2 (5.3%) with monocuspid aortic valve. Five (13.2%) TS patients had prior diagnosis of coarctation of the aorta. Four (10.5%) TS patients displayed partial anomalous pulmonary venous drainage and 3 (7.9%) showed dilatation of the ascending aorta. Four (10.5%) TS patients underwent prior cardiac surgery. Of note, some TS individuals demonstrated multiple congenital heart defects.

Twenty-two (57.9%) TS patients were classified as normal weight, 9 (23.7%) as overweight and 7 (18.4%) as obese. Five (13.2%) TS patients had arterial hypertension and 3 (7.9%) showed carbohydrate metabolism disorders. Results on karyotype and cardiovascular morbidity of TS patients are summarised in Table 2.

Echocardiography Left ventricular dimensions

Compared to controls, TS subjects displayed significantly higher values for IVSd Z score (P=0.030). Detailed information on left ventricular dimension is summarized in Table 3 for both groups.

End-systolic wall stress

No significant difference was assessed in ESWS between TS patients (n=33) and controls (n=19) in regard to different parameters indicating left ventricular wall stress (median, minimum/maximum: 125.77×103, 91.22×103/187.96×103vs. 122.98×103, 103.07×103/156.10×103 dynes/cm2; P=0.403). However, within the TS group, overweight and obese TS subjects (n=13) showed, compared to normal weight TS subjects (n=20), significantly higher values in ESWS (median, minimum/maximum: 131.43×103, 111.16×103/187.96×103vs. 123.68×103, 91.22×103/172.40×103 dynes/cm2; P=0.006). Figure 3 visualizes differences in ESWS between normal weight and overweight/obese TS subjects.

Left ventricular systolic deformation

No significant difference in GLPS_A2C, GLPS_A3C, GLPS_A4C and the subsequently calculated GLPS_AVG was found between the TS and the healthy control group. Detailed data on measured left ventricular deformation is summarized in Table 4 for both groups.

Left ventricular myocardial work and efficiency

Using the novel PSL analysis method, TS patients achieved, compared to controls, significantly higher values in global myocardial work index (BSA) (P=0.027) and global constructive work (BSA) (P=0.011). Both groups did not show significant differences in global wasted work (BSA) and global myocardial work efficiency. Within the TS cohort, heart rate correlated significantly with global myocardial work index (BSA) (r=0.558, P=0.001) and global constructive work (BSA) (r=0.445, P=0.009). Detailed results on left ventricular myocardial work and efficiency are summarized in Table 4.

Pulse wave velocity

Arterial stiffness was non-invasively assessed through PWV analysis as described in the methods. Of note, PWV analysis was not executed in three control subjects. PWV did not differ significantly between TS patients (n=33) and controls (n=16) (median, minimum/maximum: 4.80, 3.80/6.90 vs. 4.75, 3.90/6.50 m/s; P=0.661). Overweight and obese TS patients (n=13) tended to have, compared to normal weight TS subjects (n=20), higher values for PWV (mean ± SD: 5.06±0.41 vs. 4.75±0.73 m/s; P=0.174). Within the TS cohort, PWV correlated significantly with ESWS (r=0.582, P<0.001).

Discussion Increased myocardial work in TS patients

To the best of our knowledge, this is the first study that applies the novel PSL analysis method in patients with TS. Compared to controls, TS patients demonstrated a significantly higher myocardial workload according to global myocardial work index (BSA) and global constructive work (BSA) which were assessed through PSL analysis.

The elevated myocardial work, observed in the TS cohort, might be primarily explained by the cumulative overall cardiovascular morbidity linked with TS (2-4,6-8) which leads to increased left ventricular afterload and possible functional myocardial impairment.

In a recent publication of our group, we were able to demonstrate signs of significantly increased arterial stiffness in TS patients by means of abdominal aortic 2DST (6). In this study, we evaluated arterial stiffness only through oscillometric PWV which is a validated non-invasive method (19). However, no significant differences in arterial stiffness were found between both groups by means of the oscillometric PWV method used. This may be explained by the relatively young age of the population studied. In addition, oscillometric PWV analysis may not be sensitive enough to detect early manifestations of arterial stiffening in young age.

Previous studies have shown that excess weight negatively affects arterial stiffness (22,23). In this study, PWV tended to be higher in overweight and obese TS subjects, compared to normal weight TS subjects. In addition, we were able to demonstrate that overweight and obese TS subjects showed significantly higher ESWS when compared to normal weight TS subjects (Figure 3). Interestingly, PWV correlated significantly with ESWS within the TS cohort. These results might suggest that left ventricular afterload is higher in overweight and obese TS patients secondary to an increased arterial stiffness. We therefore assume that TS patients with excess weight display an even higher cardiovascular risk when compared to normal weight TS patients. Consequently, the promotion of a healthy lifestyle is crucial in cardiovascular risk management of TS.

Effects of heart rate on myocardial work in TS patients

TS is linked with an elevated resting heart rate, presumably caused by a sympathetic dysregulation (10,24,25). The examined TS subjects in this study demonstrated, compared to healthy, age-matched controls, significantly increased values for resting heart rate. Interestingly, higher values for resting heart rate were closely associated with increased myocardial work within the TS group. Opdahl et al. demonstrated that an increase in resting heart rate is related to left ventricular dysfunction and an elevated risk of developing heart failure (26). A permanent increase in resting heart rate is accompanied by higher myocardial oxygen demand which possibly promotes the process of left ventricular remodelling (26). These results might suggest the use of resting heart rate as risk stratification for left ventricular dysfunction within the TS cohort possibly leading to an earlier therapeutic pharmacological intervention in at-risk patients to prevent further cardiovascular morbidities. Further studies are required to investigate whether heart rate lowering medications (e.g., beta-blockers or funny channel inhibitors) might reduce myocardial work in TS patients.

GLPS vs. PSL

Interestingly, left ventricular GLPS was not significantly altered between TS patients and healthy controls. These results might be due to the fact that GLPS is a load dependent parameter (18). Through PSL construction, however, left ventricular afterload is considered in the left ventricular function analysis (18). In patients with increased left ventricular afterload, the myocardium must apply more work to maintain left ventricular systolic function, which is conventionally measured by EF and GLPS (18). In this case, the global myocardial work index would be increased while the GLPS would still seem to be unchanged (18). In the long-term, however, a permanently raised global myocardial work index might result in left ventricular remodelling and left ventricular dysfunction (18). According to our preliminary results, myocardial work and efficiency parameters assessed by means of the recently developed PSL analysis method may help to identify patients at risk of left ventricular remodelling and left ventricular systolic dysfunction even before abnormalities in GLPS can be detected.

Myocardial efficiency measured by global myocardial work efficiency did not differ significantly between TS patients and controls. The global myocardial work efficiency is mainly reduced by a relative increase in global wasted work compared to global constructive work. Global wasted work, however, has been shown to be primarily elevated in patients displaying heart rhythm disorders (e.g., left bundle branch block) leading to dyssynchronous movements of single myocardial segments (13). In this study, no significant differences in global wasted work (BSA) and global myocardial work efficiency were assessed between TS patients and controls. Since none of the examined TS patients presented with significant arrythmia, left bundle branch block or advanced heart failure, these results seem to be plausible. However, TS has been described to be associated with heart rhythm disorders (27-29). Further studies will have to investigate whether TS patients displaying heart rhythm disorders also show abnormalities in global wasted work and hence global myocardial work efficiency.

Limitations Study population

This study was a cross-sectional single-center study. Taking into consideration the low prevalence of TS in the general population, the TS sample size applied in this study was relatively high. However, due to the heterogenic cardiovascular morbidity of the TS cohort studied, the effects of single cardiovascular risk factors on left ventricular function are still speculative. By establishing longitudinal multi-center studies, that include larger TS cohorts, the cardiovascular pathophysiology of TS could be better evaluated.

Moreover, the examined TS cohort consisted of relatively young study participants. It therefore remains unclear whether TS patients develop more advanced left ventricular systolic dysfunction later in life and whether PSL analysis might be beneficial for risk stratification.

PSL and speckle tracking echocardiography

PSL construction is dependent on visual determination of valve opening and closure time in the apical three chamber view. In the present study, we used a relatively low frame rate of 60–90 fps. To adequately determine short time intervals (e.g., isovolumetric contraction period), higher frame rates might be required. Especially in patients with elevated heart rates, the precise visual assessment of short time intervals through valve motion is difficult. An increase in frame rate, however, might result in less accurate tracking of myocardial speckles and therefore in lower quality strain assessment and calculation. The determination of short time intervals through Doppler echocardiography might be a possible solution to assess isovolumetric time intervals more precisely. In addition, the accuracy of 2DSTE is dependent on the sonographic window and on heart rate. Excess weight, which often leads to reduced image quality, as well as an elevated heart rate, impair myocardial speckle tracking which might result in imprecise strain values.

Pulse wave velocity

In this study, PWV was assessed non-invasively through the validated oscillometric Mobil-O-Graph device. In comparison to other similar non-invasive PWV measuring devices, the Mobil-O-Graph is reported to underestimate PWV values (30). Therefore, the PWV values received in this study might be device specific and not entirely applicable with other non-invasive PWV measuring devices (30).

Conclusions

In this study, TS patients showed signs of increased myocardial workload that were only detectable by means of the novel PSL analysis method and not through conventional GLPS. In addition, elevated resting heart rate was linked with increased myocardial workload in TS subjects. The changes in myocardial work might be interpreted as an adaptative remodelling of the left ventricular myocardium in response to an abnormal left ventricular afterload and to structural as well as functional vascular impairments in TS patients.

Further studies will have to investigate whether TS patients develop advanced left ventricular systolic dysfunction later in life and whether PSL analysis might be beneficial for risk stratification in these patients. Moreover, the possible influence of interventional therapeutic medications on lowering heart rate and myocardial work in TS should be evaluated in future studies.

Acknowledgments

We would like to thank all study participants and the German Turner Syndrome Association (Turner-Syndrom-Vereinigung Deutschland e.V.) for the interest in our study.

Funding: This study was supported by the Competence Network for Congenital Heart Defects, which received funding from the Federal Ministry of Education and Research, Grant Number 01GI0601 [2014], and the German Centre for Cardiovascular Research, Grant Number 81X2800112 [2015].

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: This study was a retrospective analysis of prospectively collected data and was approved by the Ethics Committee of the Arztekammer des Saarlandes (State Chamber of Physicians of the German federal state of Saarland), Faktoreistrase 4, 66111 Saarbrucken, Germany, on March 23rd, 2018; approval statement No. 07/18. This study conformed to the standards set by the latest revision of the Declaration of Helsinki. Prior written informed consent was obtained from all patients or the parents or legal guardians of patients under legal age.

References

Cite this article as: Oberhoffer FS, Abdul-Khaliq H, Jung AM, Zemlin M, Rohrer TR, Abd El Rahman M. Assessment of left ventricular myocardial work in Turner syndrome patients: insights from the novel non-invasive pressure-strain loop analysis method. Quant Imaging Med Surg 2020;10(1):15-25. doi: 10.21037/qims.2019.09.19

## Passive and active exercises are similarly effective in ...

Sep 30, 2015 · The average age of the exercisers (68.0±5.4 yr) in their study was much lower than those of the participants in the current study (PME 83.7±4.2 yr; AME 86.0±4.2 yr). Yamauchi et al. 20 ) found an 18% improvement in AC score and 6% improvement in CS score following a 26-min home-based exercise program performed 3 days/week for 12 weeks.[Purpose] The aim of this study was to compare the efficacy of passive motion exercise and active motion exercise on functional fitness in elderly nursing home residents. [Subjects and Methods] Twenty-three (female 22 and male 1) nursing home residents (84.8±4.3 ....
From: www.ncbi.nlm.nih.gov

INTRODUCTION

It is well documented that an inactive lifestyle, such as bed rest, in otherwise healthy individuals is associated with decreased physical function exacerbated by changes in body composition, that is, decreased muscle mass and increased fat mass1). A nursing home-based lifestyle is often sedentary, and it has been reported that frail older women residing in a nursing home experienced a significant progressive loss of muscle mass2).

On the other hand, research over the past two decades clearly shows that regular exercise, that is, active motion exercise (AME), is effective for maintaining and promoting health, physical fitness, and functional independence in older adults, especially in terms of endurance, muscular strength, flexibility, and balance3,4,5). However, there are many older adults who have limitations in performing active exercises, especially weight-bearing exercises due to orthopedic conditions, poor musculoskeletal conditions, excess adiposity, poor balance, or simply age-associated sarcopenia6,7,8). Passive motion exercise (PME) may be an alternative mode of exercise for those elderly who cannot perform AME.

Machine-based PME was found to be beneficial in frail elderly users of a day care service9) as well as in community-dwelling chronic stroke patients with spastic paralysis10). However, little information is available about PME in nursing home residents, and its efficacy has not been compared with that of AME. Therefore, the purpose of the current study was to compare the efficacy of PME with that of AME in a group of nursing home-dwelling elderly people.

SUBJECTS AND METHODS

In response to a public call for participation (leaflets and oral) through proper channels, 23 residents (mean age 85.3 yr; 80 to 98 yr, 22 female and 1 male) of a nursing home in Yokkaichi City, Mie Prefecture, Japan, volunteered to participate in this study. The average nursing home residence period for these people was 8.2 years (1.9 years to 18.4 years). The participants () were then divided into a PME group and an AME group. The nursing home where the participants were residing assists only in instrumental activities of daily living (ADL)11), and all participants were able to walk indoors by themselves. No assistance was needed for these people while eating, dressing, or taking a bath. However, assistance was needed in preparing food, preparing a bathtub, preparing a shower room etc. None of them were suffering from uncontrolled hypertension, congestive heart failure, untreated ischemic heart disease, or untreated arrhythmia. None had any restrictions advised by their attending physician with regard to physical exercise performance.

Table 1.
PME (n=12)AME (n=11)
Age (yrs)83.7±4.286.0±4.2
Height (cm)149.2±6.5149.7±4.0
Body mass (kg)49.0±7.752.4±6.1
BMI (kg/m2)22.0±2.523.4±2.5
Arm Curl (times/30 sec)16.0±4.017.6±4.2
Chair Stand (times/30 sec)12.3±3.513.0±2.5
Up and Go (sec)10.3±7.28.2±1.9
Sit and Reach (cm)4.0±6.05.2±12.3
Back Scratch (cm)−12.8±12.1−10.4±11.6
Functional Reach (cm)19.3±6.515.9±5.4
12-min walk distance (m)505±209646±106
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The ethics committee of the Suzuka University of Medical Science approved the study (Approval No. 139). All participants received written and oral instructions of the study, and each gave written informed consent before participation.

PME was performed in 30-min sessions twice a week for 12 weeks with four different types of passive exercise machines (Motorcise, Combi Wellness Corporation, Tokyo, Japan). Each type of PME was performed on the respective machine for five minutes. Five minutes of warm-up and five minutes of cooldown exercises were performed before and after each session. The passive exercise machines and exercise contents are shown in . Exercises were performed on each machine for five continuous minutes, resulting in a total of 20 minutes of PME. The AME was designed based on Takeshima’s home-based resistance exercise program12), with minor modification to make it suitable for nursing home-dwelling older people. The AME was performed in 30-min sessions twice a week for 12 weeks with elastic bands (Thera-Band, The Hygenic Corporation, Akron, OH, USA). Band-based AME was performed for 20 minutes each day. Five minutes of warm-up and five minutes of cooldown exercises were performed before and after each session. In order to train all major muscle groups, band-based AME was prescribed as a combination of 3 upper body exercises and 3 lower body exercises (). Each type of exercise was performed for 1 set of 10 repetitions13) for a total period of five minutes per session. The band-based AME intensity was targeted at 13 on the Borg Rating of Perceived Exertion Scale14). The participants started with the red Thera-Band (band of lower resistance), and some of them then progressed to the green Thera-Band (band of higher resistance) during the program.

Table 2.
Machine Contents
1. Chest & Step Stepping exercise of lower limbs and simultaneous upper limb movements that simulate swinging exercise for the upper limbs during walking or jogging.
2. Shoulder & Leg Press Simultaneous upward shoulder push-pull and leg press.
3. Trunk & Twist Trunk rotation with the aim of thoraco-lumbo-sacral joint mobilization.
4. Fly & Abduction Simultaneous abduction-adduction movements of shoulder joints and hip joints as if the person is flying like a bird.
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Table 3.
ExerciseContentsRepetition
1Double arm pull-backThe elastic band is held with both hands horizontally in front of the chest in an elbow-extended position. The band is pulled back over the course of 3 to 4 seconds to simulate the breaststroke in swimming. The original position is then returned to over the course of 2 to 3 seconds. 10

2Arm curlFirst, the band is fixed and held tight under the foot, and then the other end of the band is held by the hand on the same side while resting the hand on the thigh on that side. Then the elbow is slowly bent over the course of 3 to 4 seconds to perform a full arm curl. After that, the starting position is returned to over the course of 2 to 3 seconds. This constitutes one repetition. 10

3Arm extensionThe band is held using both hands. One hand is placed on the opposite shoulder. The other hand is placed in a flexed position. Then the hand on the shoulder is slowly extended over the course of 3 to 4 seconds. The starting position is returned to over the course of 2 to 3 seconds. This constitutes one repetition. 10

4Knee extensionThe band is tied at ankle level to both legs. Both knees are at flexed in the starting position. One knee is then slowly extended over the course of 3 seconds while the other knee position remains unchanged to create resistance. The starting position is then slowly returned to, to complete one repetition. After completing the complete set, the exercise is performed for the other leg in the same way. 10

5Ankle pressThe band is held with both hands and is then stepped on by a leg in such a way that the heel of the foot makes contact with the ground but the toes are bent upward a little. Then the band is pressed slowly over the course of 3 to 4 seconds as if pushing a car’s accelerator. The starting position is returned to over the course of 2 to 3 seconds. This constitutes one repetition. After completing the complete set, the exercise is performed for the toes on the other foot in the same way. 10

6Toe liftThe band is wrapped around the distal foot including toes of one leg and then pulled diagonally to create resistance. Then the tensed band is stepped on with the other foot. After that, the toe of the band-wrapped foot is raised against the resistance over the course of 3 to 4 seconds. The starting position is returned to over the course of 2 to 3 seconds. This constitutes one repetition. After completing the complete set, the exercise is performed for the toes on the other foot in the same way. 10
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Body height, mass, BMI, and functional fitness were measured before and after the 12-wk exercise interventions in all participants. All measurements were completed within one week before and one week after the interventions.

A battery of field tests was used to assess the components of functional fitness having good test-retest reliability and validity15,16,17,18).

Upper-body strength was assessed using the 30-sec Arm Curl Test (AC)16), in which the participants flexed and extended the elbow of the dominant hand, lifting a weight (men, 8-pound [3.6 kg] dumbbell; women, 5-pound [2.3 kg] dumbbell) through the complete range of motion, as many times as possible in 30 sec (score=number of repetitions). A practice trial of one or two repetitions was given, followed by two test trials, with the best performance used for analysis.

Lower-body strength was assessed using the 30-sec Chair Stand Test (CS)16), in which the participants rose to a full standing position from a chair and then returned to a fully seated position, and continued to complete as many full stands as possible in 30 seconds (score=number of stands). A practice trial of one or two repetitions was given, followed by two test trials, with the best performance used for analysis.

Balance and agility were assessed using the 8-foot Up and Go Test (UG)16) and Functional Reach Test (FR)15). To perform the UG, the participants stood from a fully seated chair, walked as quickly as possible around a cone placed 8 feet (2.44 m) ahead of the chair, and returned to a fully seated position on the chair. The test was a timed test, and the best performance time in the test trials was recorded in tenths of a second. The participants practiced by walking through the test one time and then were timed during two test trials, with the best performance time used for analysis. To perform the FR, the participants stood with their feet together and both arms raised horizontally in front of them. They held the 0 centimeter level on the functional reach scale and then leaned forward, moving the hands forward along the scale as far possible without losing their balance (score (cm)=maximal distance the participant could reach forward beyond arms’ length). A practice trial of one or two times was given, followed by two test trials, with the best performance used for analysis.

Upper-body flexibility was assessed using the Back Scratch Test (BS)16), in which the participants placed their preferred hand behind the shoulder on the same side and the other hand behind the back, reaching up in an attempt to touch or overlap the extended middle fingers of both hands. The score was the number of centimeters the middle fingers were short of touching (negative score) or overlapped each other (positive score). A practice trial of one or two times was given, followed by two test trials, with the best performance used for analysis.

Lower-body flexibility was assessed using the Chair Sit and Reach Test (SR)16), in which the participants sat on a chair and then slowly reached forward, sliding their hands down an extended leg in an attempt to touch their toes (without bending the extended knee). The score was the number of centimeters short of reaching the toes (negative score) or reaching beyond the toes (positive score). A practice trial of one or two times was given, followed by two test trials, with the best performance used for analysis.

Cardiorespiratory fitness was assessed by performing the 12-minute Walk Test (12-MW) which assessed the maximum distance walked in 12 minutes around a 60-meter rectangular course marked into 5-meter segments19, 20). The score was the total number of meters walked in 12 minutes.

Unpaired t-tests were used for the comparison of groups before the exercise intervention. The effects of exercises were studied with repeated measures analysis of variance (ANOVA). The significance level was set at p < 0.05.

RESULTS

No significant differences at baseline were present between the PME group and AME group in any of the measured variables ().

All of the participants completed the training, and none suffered any injuries as a result of the training program. The adherence rates in the PME and AME groups were 95.8% and 93.1%, respectively.

No significant interaction (group × time) was noted in any of functional fitness variable in either group, except for the FR score (). The percentage of improvement in the FR score in the AME group was significantly larger than that in the PME group (40% vs. 9%).

Table 4.
Variables/GroupPre-test
Mean ± SD
Post-test
Mean ± SD
Change (%)Interaction (group×time)
Arm Curl (AC) (times/30 sec)
PME 16.0±4.018.8±4.8*18.9F=0.499
AME 17.6±4.219.9±4.0*14.1
Chair Stand (CS) (times/30 sec)
PME 12.3±3.513.9±3.5*15.3F=1.023
AME 13.1±2.615.4±2.1*19.2
Up and Go (UG) (sec)
PME 10.3±7.29.6±6.9*−6.1F=0.181
AME 8.2±1.97.3±1.6*−10.9
Sit and Reach (SR) (cm)
PME 4.0±6.05.4±6.5−19.7F=0.150
AME 5.2±12.35.8±10.1−62.1
Back Scratch (BS) (cm)
PME −12.8±12.1−11.3±11.7−10.7F=1.374
AME −10.4±11.6−10.6±10.710.3
Functional Reach (FR) (cm)
PME 19.3±6.521.0±6.6*9.4F=9.851**
AME15.9±5.421.3±5.1*39.9
12-min walk distance (12-MW) (m)
PME 505±209573±258*12.2F=0.224
AME 646±106727±106*13.2
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In the PME group (time effect), significant improvement was noted in the AC (19%), CS (15%), UG (6%), and 12-MW (12%) scores, but no significant improvement was noted in the SR (20%), BS (11%) and FR (9%) scores ().

In the AME group (time effect), significant improvement was noted in the AC (14%), CS (19%), UG (11%), FR (40%) and 12-MW (13%) scores, but no significant improvement was noted in the SR (62%) and BS (10%) scores ().

DISCUSSION

The purpose of the current study was to compare the efficacies of PME and AME in a group of nursing home-dwelling elderly people. In general, passive physical exercises have been reported to be less effective compared with voluntary exercises in young adults21). However, in the current study, PME was found to have a similar effect on functional fitness, except functional reach, for these very old people.

A grave change associated with aging is the progressive decline in skeletal muscle mass, a downward spiral that may lead to decreased strength and functionality8). Therefore, the European Working Group on Sarcopenia in Older People has developed a definition and algorithm for sarcopenia, with sarcopenia being defined as the loss of muscle mass and strength that occurs with advancing age8). The current study did not measure muscle mass, but improvements in AC (19% in PME and 14% in AME) and CS (15% in PME and 19% in AME) scores in very old nursing home-dwelling people may be useful in preventing or at least controlling sarcopenia in these people. The improvements in the AC and CS scores in the current study were similar to, or better than the findings from other studies of community-dwelling and apparently healthy older adults, but the improvements in the current study were achieved with much lower exercise intensities, especially in the PME group. Takeshima et al.22) found a 23% improvement in AC score and 21% improvement in CS score following a 30-min elastic band-based resistance exercise program performed on land twice a week for 12 weeks. The average age of the exercisers (68.0±5.4 yr) in their study was much lower than those of the participants in the current study (PME 83.7±4.2 yr; AME 86.0±4.2 yr). Yamauchi et al.20) found an 18% improvement in AC score and 6% improvement in CS score following a 26-min home-based exercise program performed 3 days/week for 12 weeks. The average age of the exercisers (69.2 ± 5.2 yr) in their study was much lower than those of the participants in the current study (PME 83.7±4.2 yr; AME 86.0±4.2 yr).

Fried et al.23) developed a phenotypic definition of frailty based on readily identifiable physical aspects; three or more of the following characteristics support a frailty diagnosis: unintended weight loss, exhaustion, weakness, slow gait speed, and low physical activity. Based on the above frailty definition, not all of the nursing home residents were frail, but there was no doubt that all of them need assistance to complete their ADL. However, it is obvious that they had slower gait speeds and performed fewer ADL, as represented by their UG and 12-MW performances ().

Following a 26-min home-based exercise program (age 69.2±5.2 yr) performed 3 days a week for 12 weeks20), the UG score improved by 14%, which is similar to the improvement in AME group (11%) in the current study and higher than the improvement in the PME group (6%) in the current study indicating that low-intensity AME and low-intensity PME are able to improve UG score in very old, frail elderly people.

The 12-MW score improved by 12% in the PME group and 13% in the AME group in the current study, which is similar to the improvement (16%) in 12-MW score following a 90-min aerobic training program consisting of walking outdoors 3 days per week for 12 weeks4). Although the participants of that study worked at higher intensity (70–80% of the participants’ maximal heart rate) and were apparently healthy, their mean age of 71.6 ± 7.1 yr was quite lower than those of the participants in the current study (PME 83.7 ± 4.2 yr; AME 86.0 ± 4.2 yr). Thus, the gained benefit in 12-MW score in the current study might be due to the participants having a poor initial level of physical fitness.

Improvement in lower body flexibility (20% in the PME group and 62% in the AME group) and upper body flexibility (11% in the PME group and 10% in the AME group) was satisfactory. However, it has been reported that there is large individual variation in the age-related changes in flexibility24). Another study25) reported similar results; in that study, older adults showed significant improvement in several functional fitness parameters but no improvement in trunk flexibility, which was measured by trunk anteroflexion in a sitting position with the knee extended after completion of a 12-week training program (twice a week).

Significant interaction (group × group) was noted only in the FR score, a measure of dynamic balance, in the current study. Therefore, effect of AME on dynamic balance seems to be superior to that of PME. The passive mode of training might be responsible for lesser improvement of balance. Further study is needed to overcome this limitation.

The improvements in all other measured variables except the FR score were similar in the two groups, with no significant difference. At present there is no specific recommendation for exercise volume (e.g., kcal expenditure per week, step rate, etc.) for frail and/or nursing home residents2), and as the improvements in most of the measured variables in the current study were similar for PME and AME, thus the PME could be an alternative preferred mode of exercise for very old people who cannot adhere to an AME program due to various reasons. Future studies should compare the dose-response relationship for muscle strength, especially for PME programs in elderly people.

Reports are available that describe the clinical benefits of passive exercises, for instance, passive exercise using a horseback riding machine improved insulin sensitivity and resting metabolism in diabetic patients26), and passive leg movements enhanced the tissue oxygenation level in paretic muscles of chronic stroke patients27). From these reports, PME seems to be effective in clinical settings, but the mechanisms of how exercise produced these benefits were not clearly described in those studies. Therefore, further study is needed to explore the underlying mechanisms of these benefits.

The PME machines used in the current study were designed in such a way that passive exercise could be performed automatically while maintaining adequate physiological safety for the exerciser. They automatically stop moving when the user wishes to stop exercise; that is, they do not contract the target muscle group at all. All exercises were performed in a sitting position, which further ensured the training related safety of the PME.

Regular physical activity and/or exercises are recommended for all elderly people, but many of them do not like to do exercise on a regular basis. Thus, it is very important to develop a suitable exercise menu for very old people so that they will be able to adhere to the program safely. Recently, whole body vibration exercise was found to significantly improve balance and reduce fear of falling in older adults28). Attention should be given to ensuring safety, an easy mode of performing training, and a higher intensity when designing an exercise protocol for older adults. Indeed, an important aspect in performance of exercise is to have fun, and the mode of training should be enjoyable. Tarakci et al.29) suggested that Wii-based balance therapy is effective for improving balance and walking function in children with cerebral palsy. Furthermore, studies are needed to identify adverse effects of training as well as how to increase motivation to perform exercises in frail older adults as well as in children. In conclusion, PME and AME were similarly effective in improving FF in very old people, and PME could be an alternative preferred mode of exercise for very old people who cannot adhere to an AME program due to various reasons.

## AcompÃ¡Ã±ame - Alicia Villarreal - Lyrics

AcompaÃ±ame
Porque puede suceder
AcompaÃ±ame
Que me llegues a querer
Pon tu mano, sobre mi mano
Y a tu lado, todo el mundo correre
Ven conmigo
Cierra los ojos
Y en silencio
Sin palabras
Yo mil cosas te dire
AcompaÃ±ame
Que tu amor es mi canciÃ³n
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## Cytoprotective effect of amniotic membrane extracts on ...

Background: The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts (AME) on the ocular surface exposed to benzalkonium chloride (BAC). Methods: The human corneal epithelial cell (HCEC) line SD-HCEC1s was cultured in 5 groups: normal control (NC), NC + AME, BAC, BAC + NC, and BAC + AME.Cell viability analysis, flow …Cytoprotective effect of amniotic membrane extracts on human corneal epithelial cells exposed to benzalkonium chloride in vitro.
From: aes.amegroups.com

Introduction

Dry eye disease (DED) is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles (1). Ocular irritation, stinging and foreign body sensation are symptoms frequently reported by patients which are exacerbated by substance such as benzalkonium chloride (BAC) (2).

Most of eye drops contain preservatives to prevent potential contamination, and the most common preservative used is BAC, which is present in eye drops generally ranges from 0.004% to 0.02% (3). BAC is cytotoxic to bacteria and to ocular surface epithelial cells. Therefore, there are a number of reports on the development of ocular surface disorders (OSD) in patients treated with BAC containing eye drops. BAC may induce ocular discomfort, tear film instability, superficial structure and integrity impairment, conjunctival inflammation, subconjunctival fibrosis, and epithelial apoptosis (2,4-8). The toxicity of BAC on cultivated corneal and conjunctival cells have been evaluated in many different species. It induces cytoplasmic damage and apoptosis, and induce DNA strand-breakage and mitochondrial dysfunction (4). BAC has been employed to investigate the pathogenesis of DED in animal models (9). In rabbits, topically applied 0.1% BAC twice daily over 4–14 days results in the development of DED (10,11). In mice, 0.2% BAC applied twice daily triggers clinical signs of DED including loss of conjunctival goblet cells (GCs), increased fluorescein staining, and corneal irregularity (5,12-14).

Amniotic membrane (AM) is avascular and a rich source of biologically active factors with low immunogenicity and as such, promotes healing and acts as an effective material for wound dressing. AME supports epithelialization and exhibits anti-fibrotic, anti-inflammatory, anti-angiogenic and anti-microbial features (5). In ophthalmology, AM and amniotic membrane extract (AME) are widely used for ocular surface reconstruction, including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries (15). Compared to AM, AME can be conveniently applied to the ocular surface without the risk of suture-related complications of amniotic membrane transplantation (AMT) (5). Boiled AME has also been successfully used to ameliorate dry eye induced by BAC in a murine model (5). However, the boiled avenue may destroy some bioactive factors, such as proteins may be destroyed and modified. Although there are numbers of study of BAC toxicity on human corneal epithelial cells (HCECs), there is minimal information regarding the preclinically protective effect of AME on HCECs exposed to BAC.

Based on these beneficial effects of AM on the ocular surface, we hypothesized that AME might protect the corneal epithelium from BAC-induced toxicity. To test the hypothesis, we investigated the effects of 10% AME in cultures of BAC-damaged HCECs and its possible mechanisms.

Methods Preparation of AME

The human tissues were collected and used after written consent and approval from the Second Affiliated Hospital of Guangzhou Medical University were obtained. The placent AME were obtained from healthy women with no infectious diseases including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, and with an uncomplicated pregnancy undergoing an elective cesarean to term (38–40 weeks’ gestation). The placent AME were washed with sterile normal saline and the AM was manually stripped from the chorion. After the AM was separated, it was washed 3 times in phosphate-buffered saline (PBS) with antibiotics (penicillin 50 mg/L, streptomycin 50 mg/L, neomycin 100 mg/L, amphotericin B) under aseptic conditions for approximately 15 minutes each time. After soaking in sterile saline at 2.5 mg/L for 10–20 minutes, the AM was added to an equal volume of PBS solution in the homogenizer, ground, and centrifuged at 1,000 rpm for 5 min to obtain AME. AME was acquired with a 0.22-µm filter and then stored in a −20 °C refrigerator. The protein content of the AME was measured by the Folin-phenol method.

Cell culture

HCEC lines were obtained from a corneal epithelial cell line spontaneously derived from human limbal tissue through serial culture (SD-HCEC1s) from the research group of Professor Wang Zhichong of Zhongshan Ophthalmic Center, Sun Yat-sen University (16). Thirty cell passages were utilized in this study. The cells were seeded at a density of 500/cm2 in a 6-well plate (approximately 5,000 cells/well), and cultured in a 37 °C, 5% CO2 incubator.

When the cultured cells reached 70% confluence, the culture media were removed. Cells were incubated with BAC for 1 h at concentrations of 0.02% (the concentration was determined by the inhibition concentration 50 test based on the pre-experiment). To determine whether AME has the protective effects on BAC-damaged HCECs, AME at different concentrations of 50%, 20%, 10%, 2%, 1% were added to the cultures.

The experiment was divided into the following five groups: normal control (NC), NC + AME, BAC treatment untreated group (BAC), BAC treatment withdrawal (BAC + NC), BAC treatment withdrawal + AME group (BAC + AME), and NC + AME group.

Apoptosis analysis

Apoptosis and cell death were detected by flow cytometry on floating and adherent cells using an apoptosis detection kit (V13241, Invitrogen, Carlsbad, CA, USA). The cells were collected, washed once with cold PBS, centrifuged at 1,000 rpm for 5 min, resuspended in 1× annexin binding buffer, and then incubated with Annexin V-antibody and propidium iodide (PI). After dark incubation for approximately 1 h, the final suspension was analyzed using a FACS Calibur flow cytometer (BD Biosciences, Shanghai, China) and the results were analyzed using CellQuest Pro software (BD Biosciences, Shanghai, China).

Gene expression analysis

Total RNA extraction (n=3) was performed using TRIzol Reagent (Invitrogen). The number of cells used in each group was approximately 106.The extraction method was carried out according to the instructions and quantified according to the method described previously (17). The primers were shown in Table 1. First, single-stranded cDNA was synthesized from 1 µg of total RNA using the SYBR Prime ScriptTM RT-PCR kit (DRR063S. Takara, Dalian, China), following the instructions. The mRNA expressions of caspase-8, Muc1, Muc4, Muc16, Matrix-metalloproteinases (MMPs)-1, -2, -3, -8, -9, -13 CXCL1, IL-1, and IL-6 were detected by the SYBR green system (DRR063S.Takara, Dalian, China) with real-time PCR. The cycling conditions were the following: an initial denaturation temperature of 95 °C for 2 min, then 95 °C for 10 s, then 60 °C for 34 s (fluorescence signal acquisition), then 72 °C for 30 s for a total of 45 cycles, and then lastly, a temperature increase from 60 °C to 98 °C to obtain a melting curve. The results were analyzed by the cycle threshold (CT) method and the housekeeping gene GAPDH was used as a reference.

Western blot analysis

According to previous studies (16,17), different groups of cells (approximately 107 cells in each group) were collected for western blot analysis. Cells at passage 30 were used to detect the expressions of the related proteins. The primary antibodies used were mouse anti-human caspase-8 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human Muc1 (1:100, Abcam, Cambridge, UK), mouse anti-human Muc4 (1:100, Abcam, Cambridge, UK), and mouse anti-human Muc16 (1:100, Abcam, Cambridge, UK). β-actin (1:3,000, Cell signaling, USA) was used as positive control. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO). Localization of antibodies was detected by a chemical luminescence using a ECL kit (Amersham, USA) following the manufacturer’s instructions. The images were analyzed using the Gel-Pro analyzer (Media Cybernetic, Inc.) for statistical analysis.

The expression of inflammatory cytokines by an enzyme-linked immunosorbent assay (ELISA)

ELISA (Biosource, San Francisco, CA, USA) were employed to detect the expression of MMP-1, -3, -8, -13, CXCL1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. ELISA procedures were performed following the manufacturer’s protocol.

The NC + 10% AME group was treated with IL-1β (0.1, 1.0, or 10.0 ng/mL) or TNF-α (0.1, 1.0, or 10.0 ng/mL) for 24 h to analyze the activity levels of MMP-8 protein in the supernatants of SD-HCEC1s cells. After that, the supernatants were collected and centrifuged before assays. The supernatants, pro-MMP-8 standard, and assay buffer were incubated in the 96-well plates which were pre-coated with anti-MMP-8 antibody at 4 °C for 12 hours. Then, the activity levels of MMP-8 protein were assayed using the commercial kits (Biotrak, Amersham).

Statistical analysis

All values are presented as mean ± standard deviation (SD). All analyses were evaluated with the analysis of variance followed by Boferroni’s post hoc test using IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA). In cases of nonparametric data distribution, the Kruskal-Wallis test was performed, followed by the Mann-Whitney U test. A probability value of 0.05 (P<0.05) was considered to be statistically significant.

Results AME enhances cell viability and inhibits apoptosis of SD-HCEC1s treated with BAC

As shown in Figure 1, cell proliferation assays showed that SD-HCEC1s in 10% AME possessed relatively strong growth capacities as compared to those in the 50% AME, 20% AME, 2% AME, 1% AME, and NC groups. These results showed that the SD-HCEC1s possessed a higher proliferative capacity in 10% AME after withdrawal of the 0.02% BAC.

Morphologically, the SD-HCEC1s treated with 0.02% BAC exhibited relatively more vacuolated cells (Figure 2). The SD-HCEC1s in the 0.02% BAC 1 h + 10% AME group exhibited relatively lower vacuolated cells than the cells in the 0.02% BAC group.

Flow cytometry revealed that the percentage of apoptotic cells in the 0.02% BAC, 0.02% BAC 1 h + NC, 0.02% BAC 1 h + 10% AME, NC, NC + 10% AME groups were 74.13%±4.00%, 52.60%±1.68%, 29.52%±0.72%, 20.13%±5.04%, 18.46%±4.73%, respectively. Compared to the continuously cultured 0.02% BAC group, the 0.02% BAC 1 h + 10% AME possessed fewer apoptotic cells (P<0.001, n=3) (Figure 3A,B). Compared to the NC groups, SD-HCEC1s cultured in the NC + 10% AME groups exhibited fewer apoptotic cells (P<0.001, n=3).

AME downregulates the expression of caspase-8 and enhances the maintenance of the functional properties of mucins in SD-HCEC1s

The mRNA expression of caspase-8 was significantly increased after adding 0.02% BAC to SD-HCEC1s for 1 h. However, the level of mRNA transcripts for caspase-8 was detected at lower levels in the 0.02% BAC 1 h + 10% AME group (P<0.001, n=3) (Figure 3C). The mRNA expression levels of Muc1, Muc4, and Muc16 were significantly decreased after adding 0.02% BAC for 1 h (P<0.05, n=3), while the mRNA levels of Muc1, Muc4, and Muc16 were dramatically increased (P<0.01, n=3) in 0.02% BAC 1 h + 10% AME group (Figure 4A). Western blot showed that the protein expression of caspase-8 was significantly increased in 0.02% BAC group (P<0.05, n=3). The protein expression levels of Muc1, Muc4, and Muc16 were significantly decreased after SD-HCEC1s were exposed to BAC, while the protein expression of Muc1, Muc4 and Muc16 were dramatically increased in the 0.02% BAC 1 h + 10% AME group (P<0.01, n=3) (Figure 4B,C).

To evaluate the effect of AME on the regulation of the inflammatory cytokines in SD-HCEC1s exposed to BAC, cytokines MMP-1, MMP-3, MMP-8, MMP-13, CXCL1, IL-1β, IL-6, and TNF-α were examined. Compared to the cells exposed to 0.02% BAC, the mRNA expression levels of MMP-1, -3, -13, IL-1, IL-6, TNF-α were significantly reduced in 0.02% BAC 1 h + 10% AME group (P<0.01, n=3). Interestingly, the expression levels of MMP-8 were increased in 0.02% BAC 1 h + 10% AME group (P<0.01, n=3) (Table 2). The concentrations of MMP-1, MMP-3, MMP-13, CXCL1, IL-1β, IL-6, and TNF-α proteins were significantly higher in the 0.02% BAC group than the cells cultured in both the NC group (P<0.001, n=3) and 0.02% BAC 1 h + 10% AME group (P<0.05, n=3). MMP-8 levels in NC + 10% AME were higher than the cells in NC group (P<0.001, n=3). Compared to NC group, the concentrations of MMP-1, MMP-3, MMP-13, CXCL1, IL-1β, IL-6, and TNF-α proteins in NC + 10% AME were significantly lower (P<0.001, n=3) (Table 3).

MMP-8 activity in the conditioned media was significantly reduced after treatment with 0.1, 1, and 10 ng/mL of IL-1β, respectively. Similarly, MMP-8 activity was inhibited by TNF-α. The concentrations of MMP-8 in the control group, 0.1, 1, and 10 ng/mL of TNF-α were 29.49±0.59, 22.74±1.31, 14.38±1.46, and 7.63±0.23 ng/mL, respectively (Figure 5).

Discussion

The current study demonstrated that BAC decreased HCECs viability and proliferation, and increased cellular apoptosis. Addition of 10% AME remarkably protected HCECs against BAC-induced toxicity. The key finding is that the protective effect of AME on SD-HCEC1s exposed to BAC was mediated partially by upregulation of MMP-8 and downregulation of IL-1β and TNF-α.

DED is well documented that typical pathologic alterations of dry eye involve ocular surface inflammation, conjunctival GC loss, corneal epithelium squamous metaplasia, and epithelial disruption. In DED, the inflammations may play vital roles (18,19). IL-1β and TNF-α both play important roles in the mechanisms of DED (20,21). IL-6 plays a vital role in the induction of acute inflammation which is generated by macrophages and T cells. Bian et al. found that an increase in the expressions and activities of MMPs-1, -2, -3, -8, -9, and -13 were positively associated with the severity of corneal diseases. Additionally, in corneal wound healing procedures, the generations and activities of MMPs have elevated (22-24).

BAC has been the most common preservative used in eye drops, especially in anti-glaucoma medications (6,7). BAC acts as a surfactant to solubilize ionic components into immiscible solvents, which facilitates effective emulsification and stabilization of medications, and prolongs shelf-life. Futher, BAC is superior to other preservatives in inhibiting microbial activity (3,25,26). The half-life of BAC is approximately 20 hours in the corneal epithelium and conjunctiva, which may lead to chronic ocular surface damage in patients using BAC-containing eye drops (3,5,27). Prolonged exposure to BAC caused toxicity to the ocular surface. Preservative-free eye drops are the ultimate choice for ocular surface disease. However, preservative eye drops had a risk of microbial contamination when used more than once over a period of 10 hours. It is critical to manage BAC-induced ocular surface toxicity. Our study showed that AME had beneficial effects in preventing and treating BAC-induced toxicity in HCECs (27).

AM can inhibit the release of inflammatory mediators and their matrix surface can also capture inflammatory cells, leading to anti-inflammatory effects when covering the ocular surface (15,28-30). AME, the extracted purified component of AM, has unique biological characteristics. Advantage of the application of AME is that it not only possesses the effective components of AM but also avoids the suture-related risk. It is generally believed that AME has effective components, including HA-HC complex (hyaluronic and the heavy chains of inter-α-inhibitor, which are responsible for anti-inflammatory, and anti-scarring), fibronectins, peptides, small proteins, and growth factors (15,31-33). Unlike boiled method, we employed homogenized and filtered method to acquire AME, which can possess almost all the effective components of AM maximally. This work demonstrated that AME protected SD-HCEC1s when stressed in BAC via upregulation of MMP-8 and downregulation of IL-1β and TNF-α.

In summary, this study suggested that AME provided protection for SD-HCEC1s exposed to BAC. AME maintained the normal function of SD-HCEC1s by increasing cellular activity, inhibiting apoptosis, and maintaining mucin secretion via upregulation of MMP-8 and downregulation of IL-1β and TNF-α. AME may have the potential to be employed as a topical adjunctive therapy in eyes chronically exposed to BAC.

Acknowledgments

Funding: This study was supported by the “Yangcheng Scholar” Youth Research Backbone Training Project of Guangzhou Municipal College (No. 1201581612), Guangzhou Science and Technology Project (No. 201804010038), and Guangdong Natural Science Foundation of China (2020A1515010276; No. 20 15A030313479).

Footnote

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/aes.2020.02.06). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The human tissues were collected and used after written consent and approval from the Second Affiliated Hospital of Guangzhou Medical University were obtained.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

doi: 10.21037/aes.2020.02.06
Cite this article as: Liu J, Zou H, Zeng M, Huang AM, Chen Y, Han E, Sha X, Liu Z. Cytoprotective effect of amniotic membrane extracts on human corneal epithelial cells exposed to benzalkonium chloride in vitro. Ann Eye Sci 2020;5:11.

## Association between blood urea nitrogen and 30-day ...

Introduction. Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Severe sepsis and septic shock affect millions of people around the world each year, and more than a quarter of these patients die ().The mortality rate of septic patients hospitalized in intensive care unit (ICU) is as high as 41.9% ().Association between blood urea nitrogen and 30-day mortality in patients with sepsis: a retrospective analysis.
From: apm.amegroups.com

Introduction

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Severe sepsis and septic shock affect millions of people around the world each year, and more than a quarter of these patients die (1). The mortality rate of septic patients hospitalized in intensive care unit (ICU) is as high as 41.9% (2). Death-risk stratification in septic patients enables early identification of patients at high risk of death and facilitates rational allocation of medical resources to improve outcomes. Scoring tools such as Acute Physiology and Chronic Health Evaluation (APACHE) can be used for risk stratification in septic patients, but its implementation is relatively cumbersome and can be easily influenced by some highly variable parameters such as heart rate and respiration (3). Therefore, there is a need to find clinically common and practicable markers for the rapid death-risk stratification of septic patients, which is also an important research direction of the Surviving Sepsis Campaign (4).

Blood urea nitrogen (BUN) is the main end product of protein metabolism in the human body and is excreted mainly by the kidneys. BUN level will increase when there is excessive protein breakdown or when the glomerular filtration rate decreases. Thus, BUN level can reflect protein catabolism in the human body (5) and is also a marker of renal impairment. The rate of protein catabolism increases significantly in patients with sepsis (6), and sepsis is often complicated with acute renal injury (7). These factors can lead to an increase in BUN levels in patients with sepsis. Meanwhile, the BUN test is simple and common in clinical laboratories.

Previous studies have suggested that elevated BUN level may independently predict mortality in critically ill patients (8,9); however, these studies had small sample sizes, and a large-sample study of 30-day mortality risk stratification using BUN level in patients with sepsis has yet to be performed. By using the Medical Information Mart for Intensive Care III (MIMIC-III) database, we performed this real-world study with a large sample size to investigate the relationship between BUN level and 30-day mortality in patients with sepsis. We present the following article in accordance with the STROBE reporting checklist (available at https://dx.doi.org/10.21037/apm-21-2937).

Methods Data source

The data of this retrospective cohort study were sourced from the MIMIC-III database (available at https://mimic.mit.edu/), which collects detailed information on the routine clinical care of more than 60,000 patients admitted to the ICU at Beth Israel Deaconess Medical Center (BIDMC) in Boston, Massachusetts, USA. The MIMIC-III data set is freely available to researchers around the world and has been widely used in the development of predictive models, epidemiological studies, and educational courses (10). MIMIC-III (v.1.4) contains data associated with 38,597 adult patients admitted to critical care units between 2001 and 2012 (10). All patient data were divided into different lists and organized into tables in the corresponding CSV format, including (in total) 26 tables, which are available for interested researchers to query and download (11). MIMIC-III is connected to the Social Security Database, which records other patient information, such as follow-up time and outcomes. The information related to personal privacy in the database is processed to protect privacy; for example, all the patients are anonymous and are referred to by an individual ID. The MIMIC data set does not provide a real date of admission, data of birth, or date of death; rather, it uses a mathematical model that randomly adds or subtracts the same values for the above variables. An author of this study completed a series of courses offered by the National Institutes of Health, passed the examinations, and was thus authorized to use the relevant information from the MIMIC-III database (certification No. 40764077).

Research population

The inclusion criteria were as follows: (I) all patients admitted to the ICU in the MIMIC-III database from June 2001 to October 2012, and (II) patients meeting the diagnostic criteria of sepsis according to the new criteria for sepsis (Sepsis-3); that is, an increase of 2 points or more on the sequential organ failure assessment (SOFA) in patients with suspected infection (12). The exclusion criteria were the following: (I) patients who were not admitted to the ICU for the first time, (II) patients younger than 18 years, (III) patients with incomplete BUN measurement within the first day of ICU admission or with extremely abnormal BUN levels on the first measurement, and (IV) patients with an inaccurate time of death recorded. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The MIMIC database has been approved by the Institutional Review Board (IRB) of MIT and BIDMC, both of which waived informed consent for studies related to the MIMIC-III database; therefore, our current study did not need an approval from the ethics committee of our hospital.

Variables and data

Baseline variables extracted from the MIMIC-III database included demographic features (gender, age, body mass index), type of ICU, vital signs, comorbidities, life-support measures, and laboratory test results (within the first day after admission). The type of ICU included medical ICU (MICU), surgical/trauma surgical ICU (SICU/TSICU), and coronary care unit (CCU)/cardiac surgery recovery unit (CSRU). Vital signs included heart rate, mean arterial pressure (MAP), respiratory rate, body temperature, and oxygen saturation. The comorbidities included congestive heart failure (CHF), arrhythmia, hypertension, stroke, chronic obstructive pulmonary disease (COPD), diabetes mellitus (DM), chronic renal failure, liver disease, malignancy, and coagulation disorders. Life support measures within 24 hours after admission included mechanical ventilation (MV), renal replacement therapy (RRT), and use of hypertensors. Laboratory tests included the measurements of blood white blood cell (WBC) count, hemoglobin, platelet count, blood potassium, and blood sodium.

Data were queried and extracted using Structured Query Language (SQL) with the open-source PostgreSQL (v9.6) software and its GUI software, Navicat [v.12.1.11(64-bit)-Premium].

The objective of this study was to explore the relationship between BUN level and 30-day mortality in patients with sepsis.

Statistical analysis

Normally distributed baseline measurement data are expressed as mean ± standard deviation ($x¯±s$), and the nonnormally distributed data are presented as medians and interquartile range. Count data are expressed as frequencies with percentages. Multiple interpolation was applied for missing data in very few cases. For the analysis of baseline features, statistical differences in continuous variables among five BUN groups were analyzed using one-way analysis of variance (ANOVA), and categorical variables were analyzed using chi-square test. Hazard ratios (HRs) and 95% confidence intervals (CIs) for deaths in different BUN groups were calculated using multivariate Cox regression analysis. All the covariates in Table 1 were included in the regression model for adjustment to control for confounding effects. A generalized additive model was used to identify the nonlinear relationship between BUN level and 30-day mortality. A two-segment linear regression model was created based on the smoothed curves. A segmented relationship is defined by the slope parameters and the break-points where the linear relation changes, and R package “Segmented” was used (13). Comparison of the two-segment linear regression model and the simple linear model was performed using the log-likelihood ratio test.

Statistical analyses were performed using R Statistical Software (https://www.r-project.org, The R Foundation) and Free Statistics analysis platform. All P values reported are two-tailed, and values of P<0.05 were considered to be statistically significant.

Results Study population and baseline features

A total of 20,318 septic patients admitted to the ICU were screened from the MIMIC-III database. After 5,278 patients with multiple ICU admissions were excluded, the remaining 15,040 patients who were admitted to the ICU for the first time were further screened. After 223 patients who were younger than 18 years, 2,041 patients with incomplete or extremely abnormal BUN records within the first day after ICU admission, and 63 patients with inaccurate time of death recorded were excluded, 12,713 patients were finally included in this retrospective cohort study (Figure 1). All patients completed 30-day follow-up.

The total population was divided equally into 5 groups based on the BUN level. Since all BUN measurements in the original records were integers, Q1 was defined as BUN ≤12 mg/dL, Q2 as BUN 13–18 mg/dL, Q3 as 19–27 mg/dL, Q4 as 28–43 mg/dL, and Q5 as ≥44 mg/dL. Demographic features (gender, age, body mass index), type of ICU admission, vital signs, comorbidities, life-support measures, and laboratory test results are presented as a total of all participants and by group (Table 1).

Sensitivity analysis for the relationship between BUN level and 30-day mortality in patients with sepsis

Up to 2,623 of the 12,713 patients died within 30 days after ICU admission, yielding a mortality rate of 20.6%. By calculating the HR and 95% CI of BUN level to 30-day mortality, we found in three models (without adjustment, moderate adjustment, and with all covariates included in the adjustment) that the risk of death gradually increased in groups Q2, Q3, Q4, and Q5 with increasing BUN values (all P<0.05, compared with group Q1). In the fully adjusted model II (with all covariates in Table 1 included), the risk of 30-day mortality increased by 5% for every 10 mg/dL increase in the BUN level (HR =1.05; 95% CI: 1.02–1.07, P<0.001) (Table 2). In the subgroup analyses, the risk of death increased progressively in groups Q2, Q3, Q4, and Q5 compared with group Q1, regardless of gender (male or female), age (<65 or ≥65 years), development of heart failure, MV, or use of hypertensors (all P<0.001 in trend tests; Table 3). Kaplan-Meier survival curves and log-rank test showed a significant difference in survival among groups Q1, Q2, Q3, Q4, and Q5 (P<0.001), with the shortest survival time in group Q5 (Figure 2).

Nonlinear relationship between BUN and 30-day mortality in septic patients

The multivariate Cox regression model and smoothed curve fitting revealed a nonlinear association between BUN level and 30-day mortality, and the BUN level inflection point was 41.1 mg/dL (Figure 3). Two different slopes were fitted with segmented multivariate Cox regression models. It was found the P value of the likelihood ratio test was below 0.001 (Table 4). Therefore, we used two segmented models to fit the association between BUN level and 30-day mortality. The effect value was 1.298 (HR =1.298; 95% CI: 1.224–1.376, P<0.001) for BUN <41.1 mg/dL; however, the effect value was 1.045 (HR =1.045; 95% CI: 1.016–1.075; P=0.002) for BUN ≥41.1 mg/dL (Table 3).

Discussion

In this observational, retrospective cohort study, we used the MIMIC-III database to explore the relationship between BUN level and 30-day mortality in patients with sepsis. A nonlinear relationship between BUN level and 30-day mortality was found in these patients. The 30-day mortality rate of septic patients increased as BUN level increased, and this association differed at an inflection point of 41.1 mg/dL. For BUN <41.1 mg/dL, the 30-day mortality rate of septic patients increased by 29.8% for each 10 mg/dL increase in BUN (HR =1.298; 95% CI: 1.224–1.376; P<0.001); however, for BUN ≥41.1 mg/dL, the 30-day mortality rate increased by only 4.5% for each 10 mg/dL increase in BUN (HR =1.045; 95% CI: 1.016–1.075; P=0.002). Thus, early and effective treatment is very important, and BUN can be used as a simple and quick marker for risk stratification in septic patients.

Rapid risk stratification of septic patients is valuable, especially for emergency medical service (EMS) staff. A simple and commonly used indicator can help identify patients at high risk of death early and quickly, allocate medical resources appropriately, and increase the overall success rate of resuscitation for sepsis patients. Currently, there are many tools available for risk stratification of septic patients, among which the APACHE II score is the most widely used (3). However, it involves many parameters and is cumbersome to apply, and incorporates some unstable parameters, such as heart rate and respiratory rate. Other studies have shown that indicators such as elevated plasma acetylcarnitine (14), increased width of red blood cell distribution (15), and increased neutrophil percentage to albumin ratio (16) can be used as predictors of increased risk of death in patients with sepsis. There are also studies that have used machine learning approaches to develop models for predicting the prognosis of critically ill patients, which are expected to improve early mortality prediction and support the clinical decision-making in the ICU for high-risk patients (17,18). This may become a mainstay in the future and has the potential to replace traditional disease scoring; however, there are differences among models, and the large heterogeneity limits the integration of these models. Due to the differences in the level of health care among different countries and regions, it is important to find generic and simple indicators for the rapid risk stratification of septic patients.

Organ dysfunction is a key clinical feature of sepsis (1), and acute kidney injury is a common organ injury in septic patients which increases mortality (7). According to Kidney Disease: Improving Global Outcomes (KDIGO) guidelines (19), AKI was divided into stages 1, 2 and 3, and the severity increased in turn. Correspondingly, the risk of death increased in turn. The adjusted odds ratio for in-hospital mortality was 1.679 (95% CI: 0.890–3.169; P=0.109) for stage 1, 2.945 (95% CI: 1.382–6.276; P=0.005) for stage 2, and 6.884 (95% CI: 3.876–12.228; P<0.001) for stage 3 (20,21). Common indicators used to evaluate renal function include estimation of glomerular filtration rate (eGFR), blood creatinine, BUN and so on (22). Serum creatinine and BUN are excreted through the kidneys, and both increase with the loss of renal function. However, their clinical significance differs due to their different physiological characteristics. Serum creatinine is produced by muscles. In critically ill patients, muscle proteolysis occurs and muscle content decreases, resulting in a lesser increasing extent in serum creatinine (5). Urea nitrogen is a product of protein catabolism. Critically ill patients are in a state of high protein catabolism (23), and the rise in BUN is more pronounced than that of serum creatinine (5), suggesting that BUN has additional predictive value over serum creatinine. A retrospective study from China showed that the incidence of AKI in patients with sepsis was 47.9%, and the 28-day mortality was 32.7%. BUN was an independent risk factor for death (24). Another study analyzed 398 patients with Escherichia coli bacteremia. The 30-day mortality rate was 14.1%, and the increased ratio of BUN to serum albumin (BUN/ALB) was an independent risk factor for 30-day mortality. The ROC curve of the prediction of 30-day mortality by BUN/ALB ratio was drawn, and the area under the ROC curve (AUC) was 0.712 (95% CI: 0.591–0.805; P=0.003), and the optimal cutoff value of the elevated BUN/ALB ratio was 0.3 (67.5% sensitivity and 65.1% specificity) (25). The present study also confirmed that in patients with sepsis, the 30-day mortality rate increased progressively with the increase in BUN. Filippatos et al. found higher baseline BUN to be a powerful predictor of increased postdischarge mortality in patients hospitalized for heart failure, even in the absence of severe renal failure. Even mild to moderate elevations in baseline BUN have been shown to be predictive, and the 60-day mortality was highest in patients with heart failure in the highest quartile of BUN (BUN >40 mg/dL) (26). In our present study, the BUN inflection point was 41.1 mg/dL, as shown by multivariate Cox regression modeling and smoothed curve fitting, and the mortality rate continued to rise at BUN above 41.1 mg/dL, but more slowly. An ideal biomarker for predicting prognosis should satisfy the following criteria: (I) it is produced during a pathophysiological process, (II) the change of its value can reflect the alleviation or aggravation of a specific disease, (III) it can accurately predict the risk of death, and (IV) its detection method is simple and result is stable. Obviously, BUN meets all of these four criteria in septic patients.

In 2017, an estimated 11 million people worldwide died of sepsis (27). In recent years, with the progress of medical technology, the mortality rate of patients with sepsis has decreased year by year. However, from a global point of view, there are still significant regional differences in the number of sepsis deaths, age distribution and so on. In the future, we should actively develop tools for early identification of sepsis, so as to make early diagnosis of sepsis. In addition, it is necessary to strengthen the training of guidelines for the treatment of sepsis and guide doctors to standardize treatment. Our goal is to reduce the mortality of septic patients as much as possible.

Our present study had two advantages. First, it is a real-world study with a large sample size, and all the data analyzed were naturally generated during daily patient care and thus are highly generalizable. Second, the BUN data in this study were obtained from both acute kidney injury patients and chronic kidney disease patients because preadmission renal function data are not available for emergency patients in the real-world settings in most countries, which also allows a good extrapolation of the results.

However, our study also had some limitations. First, it had a number of confounding factors due to its retrospective cohort design. Second, some of the data were missing, resulting in incomplete sample inclusion. For example, there were 2,041 patients without BUN measurements within 24 hours of ICU admission or with extremely abnormal value of the first measurement and 63 patients with the wrong time of death recorded. Third, BUN measurement was not strictly standardized in some cases. For example, errors might have occurred when BUN was measured in different hospitals and on different instruments. Finally, only septic patients were included in this analysis; for nonseptic patients, the association between BUN level and mortality may be different.

Conclusions

There is a nonlinear relationship between BUN and 30-day mortality in septic patients. The 30-day mortality rate increases with the increase in BUN level. However, when a BUN cut-off of 41.1 mg/dL is applied, the septic patients show a large difference in death risk and must be managed distinctly.

Acknowledgments

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://dx.doi.org/10.21037/apm-21-2937

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/apm-21-2937). All authors report that this article was fully funded by special funds for science and technology development of the Xinjiang Production and Construction Corps (grant number: 2016AD004) and the payments were made to Shihezi City People’s Hospital. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

(English Language Editor: J. Gray)

Cite this article as: Li X, Zheng R, Zhang T, Zeng Z, Li H, Liu J. Association between blood urea nitrogen and 30-day mortality in patients with sepsis: a retrospective analysis. Ann Palliat Med 2021;10(11):11653-11663. doi: 10.21037/apm-21-2937

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