Background: Patients with chronic obstructive pulmonary disease (COPD) are at risk for pulmonary hypertension (PH). The aim of our study was to investigate the benefit of statins for PH in patients with COPD.Methods: The study enrolled 23 million individuals from Taiwan’s population database from January 1, 2002, to December 31, 2017. COPD patients who met the inclusion criteria were enrolled, and patients with lung cancer, less than one year of observation, specific drug therapy for PH and lung transplantation were excluded.Results: A total of 643,131 COPD patients were included in the study, and only 12,308 patients developed PH during follow-up. Based on the inclusion and exclusion criteria, 8,577 PH patients were included in the cohort of patients with PH related to COPD for analysis. According to the definition of statin exposure, the final study population had 1,487 statin users and 7,090 statin non-users. The statin user group had a lower mortality related to PH than the non-user group . The mortality rate for PH in the multivariate analysis  was significantly lower for statin users than for non-users.Conclusion: Statins seem to benefit patients with PH and COPD. Pulmonary hypertension (PH) is defined as a mean resting pulmonary arterial pressure exceeding 25 mm·Hg, as evaluated by right heart catheterization. In the sixth World Symposium on PH (WSPH) update from 2018, PH is divided into five groups according to clinical presentation, hemodynamic characteristics, pathophysiology, and therapeutic strategy . PatientCurrent medical treatments for PH, including supportive therapy and new specific drugs in PH related to COPD, are lacking, and the efficacy is still uncertain; only long-term oxygen therapy can improve symptoms.Our previous study showed that the statin user group had a lower incidence rate of PH than the nonuser group  .Therefore, we further performed a nationwide, population-based, retrospective cohort study to explore the association between statins and mortality among patients with COPD and PH.The Taiwan National health insurance program, which covers 99.6% of the 23 million people in Taiwan’s population and 93% of hospitals, clinics, and pharmacies, created one of the largest and most comprehensive medical population databases in the world. We used the full population database and multiple causes of death data from the National health insurance program. The study was conducted at the Center of Health and Welfare Data Science Centers. The medical record of the full population database was used from January 1, 2002, to December 31, 2017.To assess whether the use of statins before PH diagnosis affects the study outcome, the study population was divided into statin users, defined as statin exposure within one year, and statin non-users, defined as no statin prescription within one year. This study was approved by the IRB of Kaohsiung Medical University Chung-Ho Memorial Hospital (KMUHIRB-EXEMPT(I)-20190032).The inclusion criteria were 1) age between 40 and 90 years, 2) newly diagnosed COPD between January 1, 2002, and December 31, 2015 , 3) more than one inpatient diagnosis or more than two consecutive outpatient diagnoses of COPD within one year after the primary COPD diagnosis date, and 4) treatment with COPD medications indicated in outpatient claims for more than 28 days  , and 5) The exclusion criteria were 1) COPD with lung cancer, 2) COPD with other etiologies of PH, including human immunodeficiency virus, sleep apnea, pulmonary embolism, connective tissue disease, congenital heart defect, or portal hypertension, 4) death within 28 days after COPD diagnosis, 5) specific drug therapy for PH treatment  before COPD diagnosis, 6) death within 1 year after the first PH diagnosis date, 7) lung transplantation within 1 year after the first PH diagnosis, of 8) a less than one-year observation time in the database.To resolve the immortal time bias, we set a one-year confirmation period . PatientBaseline characteristics included age, age group, sex, urbanization level, insurance premium, comorbidities, and co-medication. Comorbidities and co-medication were confirmed by medical records in the year following the PH diagnosis .To adjust for other confounders that could influence the outcome, we listed the PH treatment  in the baseline characteristics.The severity of PH is usually expressed by the WHO functional class, mean pulmonary artery pressure, or exercise capacity test, such as the six-minute walk distance. However, we could not collect those data from our database. This study used COPD severity within one year after the PH diagnosis, with oxygen therapy and heart failure (HF) used to represent PH severity. In the clinic, if patients have more severe COPD with concomitant HF, they are considered to have more severe PH. These three factors were included in the calculation of the propensity score to identify patients with the same PH severity.We used propensity-score matching to reduce selection bias. This matching method calculated a propensity score for each patient by means of dependent variables. If patients had the same propensity score, they had equal probability of developing events that we wanted to observe. The baseline characteristics of the original cohort were used to perform propensity-score matching by multivariate logistic regression. We adopted 1:1 matching to generate the statin user group and statin non-user group after follow-up.The primary outcome was PH-related mortality, which was a composite outcome that included deaths caused by PH, deaths caused by HF, and deaths caused by respiratory exacerbation (RE). Deaths were identified using the “Multiple Cause of Death Data.” The “Multiple Cause of Death Data” contains patients’ dates of death, hospitals of death, and causes of death.The all-cause mortality, deaths caused by PH, deaths caused by HF, and deaths caused by RE were secondary outcomes. The cause of death was identified by International Classification of Diseases, Ten Revision, Clinical Modification code according to the “Multiple Cause 1 to 20 (MC1-MC20)” columns from Multiple Cause of Death Data.We set a five-year observation duration to reduce the influence of switching to different statins or refusal to take statins. The follow-up period started from one year after the index date to five years after the index date. The observation time ended at death or censor occurrence. The patients in both groups who were alive or underwent lung transplantation at the end of the observation period were censored.We conducted a subgroup analysis to assess whether the association variables could affect the effectiveness of statins. Only the primary outcomes were included in the subgroup analysis.We grouped patients treated with seven kinds of statins according into the group of most commonly used statins. The seven statins were simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pitavastatin.The cDDD was the total statin exposure over the whole follow-up period. The cDDD was calculated over the entire five-year observation duration. The cDDD was divided into six levels in the user group compared to the non-user group: non-user, cDDD <28, cDDD ≥28 but <90, cDDD ≥90 but <180, cDDD ≥180 but <365, cDDD ≥365 but <730, and cDDD ≥730.The duration of statin use was calculated by year and divided into six levels in the user group compared to the non-user group: non-user, <0.5 year, ≥0.5 year but <1 year, ≥1 year but <2 years, ≥2 years but <3 years, and ≥3 years.The intensity of statin use was calculated by dividing cDDD by the duration of statin use over the whole five-year observation period. Then, statin use was divided into four categories to determine whether the intensity could affect the protective effect of statins: non-user, intensity <10, intensity ≥10 ≤ but <20, intensity ≥20.The observation times for the sensitivity analysis were 1 year, 3, 5, 7, 9 years, and the end of the database to detect the effects of different observation durations.For baseline characteristics, continuous variables are presented as the means (standard deviations), and categorical variables are presented as percentages. Continuous variables were analyzed by Student’s t-test or analysis of variance, and categorical variables were analyzed by Fisher’s exact test or chi-square tests if numbers were less than 30.The mortality of PH was assessed using a time-dependent Cox proportional hazard model to estimate the hazard ratio between the two groups. The multivariate model was adjusted for demographic characteristics, sex, comorbidity, co-medication, and COPD severity. Because of potential interactions and collinearity between characteristics, stepwise selection was performed to select the important factors to construct the regression model. All the above analyses were performed using SAS 9.4. software. The statistical significance was determined with two-tailed analyses and α = 0.05.A total of 643,131 COPD patients were included in the study, and only 12,308 patients developed PH during follow-up. Based on the inclusion and exclusion criteria, 8,577 PH patients were included in the PH related to the COPD cohort for analysis. According to the definition of statin exposure, the final study population had 1,487 statin users and 7,090 statin non-users. p < 0.001). The age distributions were similar between groups, with most patients over 60 years old and the majority being male. The insurance premium showed the same distribution, with more patients paying below 22,800 Taiwan Dollars and a significant difference between the two groups (p = <0.001).The PH cohort contained 1,487 statin users and 7,090 non-users. The mean age of the patients in the user group (72.52 years) was lower than that of the non-user group , coronary artery disease (p < 0.001), and ischemic stroke (p < 0.001). The proportions of most comorbidities were significantly higher in the user group than in the non-user group. There was no significant difference in chronic liver disease, arrhythmia, interstitial pulmonary diseases, asthma, malignancy, hemorrhagic stroke or left ventricular hypertrophy between groups.Because of the indications for statins, patients with statins had a significantly higher prevalence of dyslipidemia (p = 0.007), In addition, there were no significant differences in oxygen therapy or specific drug therapy between groups. Regarding medication for COPD, more than half of the patients used methylxanthines. Regarding the severity of COPD, there were significant differences in both severe and moderate exacerbations (p < 0.001) between the two groups. The distributions of severe and moderate exacerbations showed that most patients had no exacerbation in the firth year after COPD was diagnosed, and the second highest number of patients had over two exacerbations. The detailed baseline characteristics of the PH patients before matching are presented in Co-medication, except with digoxin, had the same trend for comorbidity. More patients in the non-user group used digoxin . After matching, the propensity scores in the two groups were similar, with no significant differences .To identify similar patients in the control group, the study population was subjected to 1-to-1 propensity-score matching, and age group, sex, insurance premium, urbanization, hypertension, diabetes, HF, asthma, oxygen therapy, specific drug therapy and COPD severity were used to calculate the propensity score. Before matching, the propensity scores of the user and non-user groups were significantly different . Regarding the secondary outcomes, most patients with mortality related to PH died due to RE , and few patients died due to PH or HF.Regarding the primary outcome, during the five-year observation duration, 462 (34.87%) statin users and 647 (48.83%) statin non-users died, among whom 145 (10.94%) statin users and 210 (15.84%) statin non-users died of causes related to PH. Regarding the primary outcome, the total follow-up times of the user and non-user groups were 3,751.18 and 3,784.92 person-years, respectively. The statin user group had a lower mortality related to PH than the non-user group  and multivariate analysis  of mortality related to PH both showed significantly lower mortality for statin users than non-users. The above results suggested that statins could reduce the risk of mortality related to PH by approximately 22–30% and that they were effective in patients with PH related to COPD. Regarding secondary outcomes, there was significantly lower risk for deaths caused by PH or HF  and all-cause mortality .The Cox proportional hazard model analysis of primary outcome and secondary outcome of PH patients is summarized in p < 0.001). In the multivariate analysis, all baseline characteristics were included in the adjusted model. When statin users were over 70 years old, were male, had lower insurance premiums, had comorbidities including chronic kidney disease, interstitial pulmonary disease and HF, and were on oxygen therapy, they had a significantly higher risk of mortality than nonusers. Regarding COPD severity, patients with moderate exacerbation had a significantly higher risk than those with no moderate exacerbation. Furthermore, there was a trend in severe exacerbation in that patients with more severe exacerbations had a higher risk of PH  statin users taking this statin. The least commonly used statin was pitavastatin. The Cox proportional hazard model analysis of all-cause mortality in different kinds of statins is summarized in p = 0.044) and rosuvastatin  had a significantly lower risk of mortality. Among all statins, lovastatin had the lowest mortality rate .All of the statins showed a trend toward low risk of mortality (crude HR < 1.00). Patients using lovastatin . Additionally, patients with a cDDD above 365 had dose-dependent response. The higher the cDDD was, the lower the risk of mortality would be .The cDDD was calculated as total statin exposure standardized by DDD over the whole follow-up period. There were six levels of classification for cDDD, and a multivariate Cox proportional hazard model analysis was performed. The statin non-user group was a reference in the analysis. Forty percent of statin users had a cDDD of more than 730. p = 0.015). This result suggested that statin use was effective against PH when the duration of use was longer than one year.The duration of statin use was calculated by year and divided into six levels. As shown in p trend=<0.001). Patients whose statin DDD was above 10 per month started to have a significantly lower risk of mortality . This result suggested that statins had a protective effect against PH until the patients the DDD exceeded 10 per month. The intensity of statins was calculated by dividing cDDD by the duration of statin use over the entire five-year observation period. Then, the patients were divided into four levels, and multivariate Cox proportional hazard model analysis was performed. The normal DDD per month of statins is 30. As presented in p < 0.001) After adjustments for risk factors in the Cox proportional hazard model analysis, the results maintained a significantly lower risk of PH mortality. These results suggest that statins may decrease the risk of mortality from PH by 22% and show a benefit for PH patients.In our study, statins were effective in improving PH outcomes in COPD. Regarding the primary outcome, the five-year cumulative mortality was 10.94% for statin users and 15.84% for statin non-users. The PH patients with the statin user group had a lower mortality rate than those in than the non-user group  and exam results in the NHI database, it was difficult to confirm COPD severity in patients. We used the assessment of exacerbation risk in the Global Initiative for Chronic Obstructive Lung disease guidelines to define the severity of COPD. The assessment tool is divided into high-risk and low-risk groups according to the number of exacerbations over the previous year. Severe exacerbations are exacerbations leading to emergency room or hospital admission, and moderate exacerbations are exacerbations were treated with SABA plus antibiotics or oral corticosteroids. If patients had ≥2 moderate exacerbations or ≥1 severe exacerbation over the previous year, they were considered have a high exacerbation risk. If patients had no exacerbations or one moderate exacerbation, they were considered to have a low exacerbation risk . The sevBecause of the pathogenesis of PH involves proliferation of lung vasculature, vasoconstriction, and thrombosis, the anti-proliferative effects and antithrombotic effects of statins as a RhoA/Rho-kinase inhibitions seem to be benefic on PH .in vitro studies, the efficacy of statins in clinical trials is still unclear. Each statin seems to have a different contribution to protective effect and effectiveness. Compare to pharmacokinetic and pharmacodynamics property of each statin, they were no consistent in bioavailability, protein binding, lipophilicity, half-life, and other metabolism properties. Besides, we could not find studies about comparing the effects of anti-proliferative and anti-inflammatory or the potency of RhoA/Rho-kinase inhibition. Many randomized controlled trials support the benefit of statins in patients with PH related to COPD. In 2009, Lee et al. performed the first randomized controlled trial to explore the effectiveness of statins in this population. After using pravastatin 40 mg one per day for six months, the statin users showed significantly improved exercise tolerance and decreased PH and dyspnea during exercise compared to the placebo group (p < 0.001). Recent studies of simvastatin, rosuvastatin, and atorvastatin showed that they consistently and significantly improved 6 min walking distance results and directly reduced pulmonary artery pressure  and deaths caused by PH or HF  were a significantly lower in the statin user group than in the non-user group. Although most patients died due to RE, the findings was only a trend of lower risk, with no significant difference. The distribution of causes of death was similar to the most common symptoms that caused patient hospitalization in the Chen et al. study. The majority of patients were hospitalized for dyspnea (93.5%), cough (89.8%), and sputum (79.7%) rather than symptoms caused by PH or HF, such as edema (8.2%)  .In previous randomized controlled trials, although statins showed some benefits for PH, the assessments of lung function and heart function, including cardiac output, right ventricular size, FEV1, and lung capacity, did not show significant differences compared to the placebo groups. Only the findings of Chogtu et al.’s study, which showed a significant reduction in COPD exacerbations, are consistent with those of this study . Death cIn our study, the most commonly used statin was atorvastatin, with nearly 40% of users taking this statin, and the least used statin was pitavastatin, because pitavastatin was the most recent statin to be included in the National health insurance program. Most statins had a trend toward reducing the incidence of PH incidence and the risk of mortality; however, patients in part I who used only pravastatin and atorvastatin had a significantly lower incidence, and patients in part II using lovastatin and rosuvastatin had a significantly lower risk of mortality. The results revealed that the statins seem to have a differing levels of protection and efficacy. When the pharmacokinetic and pharmacodynamic properties of each statin are compared, it becomes apparent that the statins are not the same in terms of bioavailability, protein binding, lipophilicity, half-life, or other metabolic properties. In addition, we could not find studies comparing the antiproliferative and anti-inflammatory effects or the potency of RhoA/Rho-kinase inhibition by these statins.The probable reason for the inconsistent statin effect between incidence and mortality is that some patients switched to another statin during the observation duration. Therefore, it was difficult to distinguish which statins contributed to the effect. Another reason is the definition of statin grouping. The study divided patients into each statin group according to the statin with the highest cDDD during the observation duration. Perhaps the first statin that the patient was exposed to after the index date or the most recent statin treatment relative to the events of the patient have more influence on the effect. Until now, randomized controlled trials have been performed for only pravastatin, simvastatin, atorvastatin, and rosuvastatin to explore their effects on PH. More direct or indirect comparative studies of statins are needed to support these results.In the subgroup analysis, the results of two parts showed a dose-dependent effect and time-dependent effect. When patients had a higher cDDD and longer duration of statin use, they had a lower risk of PH and mortality related to PH.p = 0.001).To understand the association between cDDD and the duration of statin use, a subgroup analysis of intensity was performed. The intensity indicates the cDDD of statins per month. The normal cDDD per month is 30. However, the DDD is an average maintenance dose per day, so it may represent a moderate intensity of statin use according to their lipid-lowering effects . PatientThe dose-dependent and time-dependent effects of statins are common to many studies in different populations, making the observed effect more robust . In summTo our knowledge, this study is the first to explore the protective effect of statins against PH in COPD patients. Moreover, it is the first population-based cohort study to explore the protective effect and efficacy of statins in COPD patients. There are two strengths in our study. First, the NHI database is one of the largest and most comprehensive medical population databases in the world. The data can be extracted from 1995 to 2017 and cover 99.6% of the 23 million people in the Taiwan population. The study had a large sample size, even after propensity-score matching was conducted. There were 41,163 COPD patients and 1,325 patients with PH related to COPD in the final analysis of the study. In contrast to that of other studies, the sample size of patients with PH related to COPD is only 40 to 60 patients . In geneIn addition, RCTs include only more stable PH patients and relatively healthier patients. Chogtu et al. included patients with COPD who were stable for at least 2 weeks and excluded patients with asthma and cardiac disorders such as arrhythmias and unstable angina . Our stuThis study has several limitations. The disease diagnosis and severity assessments of the morbid conditions may not accurately reflect the real situation in this study that could have influenced the outcome of the measurements. The absence of information relating to other factors such as cigarette smoking cannot be underestimated. The study population and outcomes were all defined based on the International Classification of Diseases, Ninth Revision, Clinical Modification or International Classification of Diseases, Ten Revision, Clinical Modification in the admissions record and not on clinical diagnosis because the National health insurance database lacks examination results and lab data. Although we used multiple definitions, such as combined treatment or examination, to improve accuracy, uncertain error is still expected. The limitation of the National health insurance database also leads to the study not having access to smoking status, lifestyle, lung function, or PH severity. Therefore, adjustments could not be made for these important factors or potential confounders, which may influence the outcomes.Our study suggests that statins have protective effects and efficacy against PH related to COPD and can reduce the mortality of PH. Moreover, the benefits of statins are dose dependent and time dependent.Age over 60 years, male, low income, HF, and COPD severity are risk factors for mortality, and chronic kidney disease and interstitial pulmonary disease are risk factors for mortality among PH patients.


Elevated levels of alanine aminotransferase (ALT) were the lowest in the MD group (p = 0.011) along with resistance to insulin (homeostasis model assessment-insulin resistance (HOMA-IR)), which was highest in the ND group (p = 0.001). Chronic consumption of alcohol was independently and inversely associated with fatty liver and insulin resistance after adjusting for obesity, hypertension, fasting hyperglycemia, habit of drinking sweet beverages, physical activity, and age . We found that regardless of the type of alcoholic beverage, chronic consumption of alcohol is inversely associated with insulin resistance and fatty liver in Japanese males. This study had limitations, most notably the lack of investigation into diet and nutrition.We aimed to elucidate the effect of chronic alcohol consumption on fatty liver. We assessed the consumption of alcohol in 2429 Japanese males (mean age: 54.2 ± 9 years); they were classified according to average consumption into non-drinkers (ND), light drinkers (LD), moderate drinkers (MD), and heavy drinkers (HD). The prevalence of fatty liver was the lowest in the MD and highest in the ND group ( Alcohol liver disease (ALD) is a major cause of liver disease worldwide. Excess alcohol consumption is reported to lead to ALD, and 90% percent of people consuming more than 60 g of alcohol per day have been shown to develop steatosis . Non-alcIn this study, we investigated the relationship between the consumption of alcohol and the prevalence of fatty liver and resistance to insulin in Japanese men. Our cross-sectional study used data from 2461 males (age range: 27–89 years) who had consecutively undergone comprehensive annual examinations for employee or resident health check-ups at Nara Health Promotion Center during 2012. Most subjects were asymptomatic. Of these, 32 were excluded from the study for being positive for the hepatitis B surface antigen or anti-hepatitis C virus antibody. The remaining 2429 participants  were included in this study. This study was approved by the Ethics Committee of Nara Medical University.2). Obesity was defined as a BMI of ≥25 kg/m2 according to the criteria established by the Japan Society of Obesity. Waist circumference (WC) was measured at the level of the umbilicus. All subjects were examined following a 12-h overnight fast. Sitting blood pressure was measured using an automatic sphygmomanometer. Hypertension was defined as a systolic blood pressure (SBP) of ≥140 mmHg, a diastolic blood pressure (DBP) of ≥90 mmHg, or current use of antihypertensive medications.The information collected using a standardized questionnaire included alcohol consumption, habits of drinking sweet beverages and eating greasy meat, and physical activity . The bodAll biochemical analyses were performed using standard techniques. Fasting hyperglycemia was defined as a fasting blood glucose of ≥110 mg/dL or current use of antidiabetic medications. An elevated level of alanine aminotransferase (ALT) was defined as ≥ 30 IU/L. Resistance to insulin was assessed using the homeostasis model assessment-insulin resistance (HOMA-IR), which was calculated as the fasting insulin (μU/mL) × the fasting glucose (mg/dL)/405, and was defined as ≥2.5. According to the Japanese criteria, metabolic syndrome was defined as a WC of ≥85 cm and the presence of two or more of the following risk factors: medicated for hypertension, SBP of ≥130 mmHg or DBP of ≥85 mmHg; medicated for dyslipidemia or high-density lipoprotein (HDL) cholesterol of <40 mg/dL and/or triglycerides of ≥150 mg/dL; and medicated for diabetes or fasting blood glucose of ≥110 mg/dL.Fatty liver was identified by ultrasonography when increased liver echogenicity (“bright liver”) and a contrast between the hepatic and renal parenchymal tissues (“liver-kidney contrast”) were recognized. Ultrasonography was performed using the LOGIQ 7, with a 4-MHz convex array transducer .Each subject reported their frequency of consumption of alcohol per week, as well as the types and volume of alcohol consumed per day for the last year. The consumption of alcohol in grams was calculated based on the information provided, by using the representative percent of alcohol for each type of alcoholic drink: 5% for beer, 15% for sake, 25% for shochu, 40% for whiskey, and 12% for wine. To calculate the average consumption of alcohol per day in grams of ethanol, the frequency of the consumption of alcohol per week was multiplied by the grams of ethanol per day and divided by 7 days. Subjects were then classified according to their consumption of alcohol: non-drinker (ND), 0 g/d; light drinker (LD), <30 g/d; moderate drinker (MD), 30–60 g/d; and heavy drinker (HD), ≥60 g/d.Data were expressed as the mean ± standard deviation. Continuous variables were compared using a one-way analysis of variance, and the significance of individual differences was evaluated by using the Bonferroni test if the ANOVA was significant. Categorical variables were compared using the chi-square test.p-value of <0.05 was considered to be statistically significant. All calculations were performed using the SPSS software .Multivariate logistic regression analyses were used to assess whether the categories of consumption of alcohol were independently associated with the prevalence of fatty liver and resistance to insulin. A p < 0.001). Similarly, the levels of aspartate aminotransaminase (AST), gamma-glutamyl transferase (GGT), and triglycerides were also significantly increased across the categories with an increasing consumption of alcohol . However, we observed a U-shaped association between the level of ALT and the categories of consumption of alcohol. Respectively, HOMA-IR was noted to be significantly higher in the ND group than those in the LD, MD, and HD groups. The prevalence of metabolic syndrome was noted to be the lowest in the LD group and highest in the HD group. The adjusted standardized residuals in the ND, LD, MD, and HD groups were −0.6, −2.8, 1.0 and 4.2, respectively. The percentages of the use of lipid-lowering medications or antidiabetic medications were not significantly different among the four groups.The characteristics of the study population are shown in p < 0.001). There was a U-shaped association observed between the prevalence of fatty liver and the categories of consumption of alcohol. The adjusted standardized residuals in the ND, LD, MD, and HD groups were 4.2, −2.8, −1.9 and 0.2, respectively (2) was not significantly different among the four groups (p = 0.133) (p = 0.011). The adjusted standardized residuals in the ND, LD, MD, and HD groups were 2.6, −1.8, −2.0 and 1.2, respectively. Similarly, we noted a U-shaped association between the prevalence of elevated levels of ALT and the categories of consumption of alcohol (p = 0.001). The adjusted standardized residuals in the ND, LD, MD, and HD groups were 3.9, −1.9, −2.4 and −0.2, respectively. Accordingly, there was a U-shaped association shown between the prevalence of the resistance to insulin and the categories of consumption of alcohol  B. The pr alcohol C. The pr alcohol D.Chronic consumption of alcohol was demonstrated to be independently and inversely associated with fatty liver after adjusting for age, obesity, hypertension, fasting hyperglycemia, habit of drinking sweet beverages, and physical activity. The odds ratios were shown to be as follows: ND, 1; LD, 0.682; MD, 0.771; and HD, 0.840. Light consumption of alcohol exhibited the most decreased odds ratio . Similarn = 62, 6.9%) when compared to all drinkers  . The BMI was not shown to be significantly different among the ND group and various types of drinkers. Similarly, the level of ALT was not observed to be significantly different among the ND group and various types of drinkers; however, ALT tended to be the highest in the ND group. On the other hand, the level of GGT was observed to be the lowest, whereas HOMA-IR and the prevalence of fatty liver were reported to be the highest in the ND group. The percentage of the use of antidiabetic medications was not significantly different among the ND group and various types of drinkers; however, it tended to be higher in the ND group  . The pre9, 5.8%) .This study showed that consumption of alcohol is inversely associated with insulin resistance and fatty liver. We observed a U-shaped relationship between the consumption of alcohol and the prevalence of fatty liver and elevated levels of ALT. Similarly, we observed the same relationship between the consumption of alcohol and the prevalence of elevated resistance to insulin. The prevalence of fatty liver and resistance to insulin was shown to be the lowest in the MD group, whereas they were observed to be the highest in the ND group. The percentage of those who often drink sweet beverages was significantly decreased in association with alcohol consumption, and the percentage of physical activity was significantly higher in the LD and MD groups than those in the ND and HD groups. To avoid potential cofounders, we performed a multivariate logistic analysis. We demonstrated that chronic consumption of alcohol was independently and inversely associated with fatty liver and resistance to insulin after adjusting for obesity, hypertension, fasting hyperglycemia, habit of drinking sweet beverages, physical activity, and age. Moreover, regardless of the type of the alcoholic beverages, HOMA-IR and the prevalence of fatty liver was shown to be lower in the various types of drinkers compared with the ND group. Excessive consumption of alcohol is known to be harmful and has been associated with cancer, cardiovascular diseases, type 2 diabetes, liver cirrhosis, and stroke ,18. In cSome reports suggest that moderate consumption of alcohol has a protective effect against the development of resistance to insulin ,27. Our In the present study, GGT and AST were increased across the categories of alcohol consumption, whereas there was a U-shaped association between the level of ALT and the categories of alcohol consumption. GGT, ALT, and AST are the most frequently used markers for the early detection of alcoholic liver disease (ALD). GGT is usually higher in patients with ALD compared to those who have other liver diseases. An elevation of AST is observed in ALD, and ALT levels are commonly lower than AST. However, all these laboratory values are not specific for ALD, and they have low sensitivity and specificity. ALT has the lowest sensitivity among them . The levIt has been reported that modest consumption of wine is associated with a reduced prevalence of NAFLD . HoweverOur study had some limitations. First, we did not enroll female subjects. Although 2146 female subjects were investigated, 1620 (75%) subjects did not drink, and the number of those who drank more than 20 g/d was only 172, while the number of subjects with fatty liver was only 24 among those subjects. Therefore, the sample size was insufficient to elucidate the association between chronic consumption of alcohol and fatty liver in women by the methods used in the present study. Second, this study was a cross-sectional study. Third, as hepatic steatosis was diagnosed by ultrasonography, there was limited accuracy for the detection of mild steatosis. The assessments of liver were performed by ultrasound which bears limitations such as the detection of hepatitis and fibrosis. Fourth, most of the subjects were asymptomatic and able to engage in their work. The results of this study might, therefore, not be applicable to individuals who have liver fibrosis or other liver diseases. Fifth, the subjects only provided their habit of drinking sweet beverages and eating greasy meat. We did not further investigate their diet or nutrition, especially fructose and saturated fatty acids, which have been shown to play a pivotal role in the development of fatty liver. Sixth, the study was conducted on the normal-weight Japanese population, which is very different from that of western countries, where fatty liver occurs in overweight or obese individuals with insulin resistance and often with diabetes, and where the most prevalent consumption of alcohol is represented by wine. Seventh, the subjects only provided their consumption of alcohol for the last year. We did not investigate former drinkers and lifetime drinking history.In conclusion, chronic consumption of alcohol is inversely associated with insulin resistance and fatty liver in Japanese males. However, the results demonstrated in the present study do not suggest the safeness of alcohol consumption for everyone. Thus, the threshold for harm induced by the consumption of alcohol remains unclear.


The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening. The human body consists of highly sophisticated biological systems . Cells fCells in the natural environment are embedded in the extracellular matrix (ECM), forming a complex three-dimensional (3D) structure . The ECMA number of 3D culture systems are already available . Biomate2, nutrients, waste and soluble factors [Hydrogels have 3D structure, hydrophilic property, and polymeric networks capable of absorbing large amounts of water or biological fluids . These h factors . Therefo factors ,14. Hydr factors . Recentl factors . Similar factors .Natural hydrogels are made up of natural substances such as collagen, alginate, hyaluronic acid and many more that promote several cellular functions with a range of endogenous factors present, which can benefit the viability, proliferation, and differentiation of many cell types ,51,52,53Escherichia coli to produce animal-free HA [Hyaluronic acid (HA) is distributed in many tissues, such as skin and cartilage . HA can -free HA . The rol-free HA ,73,74. I-free HA ,76. Tumo-free HA . Other r-free HA . Another-free HA . Häckel -free HA . Further-free HA . Recentl-free HA . Finally-free HA .Alginate is derived from the cells of brown algae, and its monomers have the ability to cross-link to form hydrogels . NormallSolid scaffold-based cell culturing is one of the older techniques used in the field of 3D cell culture . In this2 can be used as the gas foaming agent, and the porosity of the scaffold can be controlled by the amount of gas dissolved in the polymer. The melt molding method uses both polymer and porogen, which are poured into a mold and heated above the polymer glass transition temperature [Porous foam-solid scaffolds have high porosity and a uniform interconnected structure. Many attempts have been made to fabricate porous foam-solid scaffolds ,109. Parperature ,114. Varperature ,117,118.perature . Human hperature ,121.Fibrous scaffolds provide a large surface area for cell growth in 3D cell culture . These sAnother way to fabricate a porous scaffold is 3D printing. This method allows for easier and more detailed manufacturing than the above two methods and allows various architectures and control of mechanical stability . The metOne of the ideal scaffolds is a decellularized matrix that provides natural geometric morphology, flexibility, and mechanical strength, which is difficult to mimic perfectly with synthetic scaffolds. Recently, various approaches have been introduced to fabricate decellularized scaffolds, including perfusion of the whole organ (recommended for dense organs/tissues), application of a pressure gradient (employed for hollow tissues), use of supercritical fluid (appropriate for long-standing storage of decellularized scaffolds), and immersion and agitation (suitable for thin tissues) ,156,157.Cell culture plates can be covered with biomaterials with low cell-binding properties to prevent the cells from adhering to the surface. This method is one of the older techniques to generate self-assembled cellular structures in media for 3D cell culture. This system inhibits the attachment of cells to the surface of the culture plate, resulting in force floating of cells. Force floating improves cell-to-cell interactions, enabling multicellular aggregation. To provide attachment-resistant cell surfaces, cell culture plates or surfaces are coated with polymers that possess low cell-binding properties, such as 2-hydroxyethyl methacrylate (poly-HEMA), polyethylene glycol, chitosan, agar, and agarose ,171,172.Three-dimensional (3D) cell culture has several applications in the field of biological science. These applications can be divided into three categories: cancer research, stem cell culture, and drug toxicity and screening. With the growing demand for effective validation in the field of oncology, there is a demand for research tools that provide clinically significant results for safety and efficacy testing of anticancer drugs. Moreover, 3D culture techniques have been able to produce 3D models, such as oncospheres, spheroids, and other tumor models that closely resemble the natural tumor microenvironment due to the proper supply of nutrients, oxygen, and intercellular interactions ,182. IncAnimal models and 2D culture systems have been a standard to determine anticancer drug effects on growth inhibition and apoptosis. However, the results produced from these systems showed relatively low similarity in clinical outcomes ,184. OwiCancer cells grown and maintained in 3D models show phenotypic heterogeneity . This isRecently, stem cells have become an important research tool in biology, medicine and toxicology, leading to a rapidly developing new scientific field. The importance of stem cells is evidenced by the increasing number of articles published each year. This rapid development requires new means of providing high-quality, well-defined and scalable techniques for the generation of stem cells. It is essential to proliferate stem cells to be used as a tool in scientific disciplines. Embryoid bodies (EBs) are 3D aggregates generated in suspension by pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) . EB diffOne of the major efforts in the stem cell field is to develop therapeutic applications for the regeneration of diseased, dysfunctional or complex injured tissues. To that end, it is essential to secure technology for stem cell proliferation with increased cell viability and differentiation potential . VariousThree-dimensional (3D) cell culture has emerged as a promising method for the generation of organoids that could serve as a model to study various disease mechanisms or the toxicity of new drugs . OrganoiHerein, we introduced 3D cell culture methods and summarized the trends in the application of this technology. The 3D cell culture technologies have already emerged as an important tool that can facilitate research in the biomedical field with more realistic results. However, 3D culture methods have many problems to overcome. First, there are no standardized culture methods. The 3D cell culture method is more difficult and more complicated than that of 2D cell culture. Thus, it has low reproducibility and requires a high level of skill. The second problem is that 3D cell culture is still inappropriate for completely simulating the intrinsic properties of cells. One example is a vascular problem. In the body, tissues are constantly supplied with nutrients and oxygen from microvessels in the tissues. Cells cannot survive beyond 150 μm from a microvessel. Thus, angiogenesis is an important consideration for successful 3D cell culture. When tissue is regenerated in vitro, the 3D tissue should be supplied with nutrients and oxygen around it until blood vessels grow into the tissue. In this case, growth factors as well as vascular endothelial cells and stem cells may be used to promote angiogenesis. However, the problem is that necrosis can occur before blood vessels are formed due to the insufficient supply of nutrients and oxygen within the 3D tissue. Finally, even if this simulation is correctly achieved, there is still a lack of research on analytical methods for verification. Most analyses now depend on visual images. Therefore, it can be difficult to clearly judge the standards and grasp the characteristics inside 3D structures. For this reason, standardization of the analysis of 3D cell culture is still difficult. Although there are still many problems to overcome in 3D cell culture, the introduction of advanced 3D cell culture technologies has attracted several researchers to shift their attention from 2D to 3D cell culture systems. Thus, 3D cell culture technology is expected to contribute greatly to future research with the potential to create excellent outputs.


In comparisons among exercises, the path length and average velocity variables showed a significant decrease in the eye-opened condition, and the path length variable showed a significant decrease in the eye-closed condition (p < 0.05). In muscle activation, CE showed a significant increase in the gluteus medius (Gmed) and decrease in the rectus femoris (RF), tibialis anterior (TA), and RF/biceps femoris (BF) ratio in the eye-opened condition compared to the baseline and a significant decrease in RF and RF/BF ratio in the eye-closed condition (p < 0.05). Both CE and CSE improved the static balance ability. Furthermore, muscle activation significantly increases in Gmed and decreases in the RF, TA, and RF/BF ratio. Therefore, we recommend including CE in an exercise program that has the purpose of improving static balance ability.Balance ability is a necessary exercise factor required for the activities of daily living. This study investigated the effects of short-term complex exercise (CE) and core stability exercise (CSE) on balance ability and trunk and lower-extremity muscle activation on healthy male adults. Twenty-nine healthy male adults were included. All performed CE and CSE for 1 min each; the exercise order was randomized. The primary and secondary outcomes were balance ability and muscle activation, respectively. In balance ability, CE showed a significant difference in all variables in both eye-opened and eye-closed conditions compared with the baseline ( Balance ability is essential in performing activities of daily living (ADLs) such as walking, sitting, and standing as well as sports activities. Balance training is considered necessary for ADLs as it facilitates improved work ability in standing and sitting positions and prevents the elderly from falling . FurtherIn 2010, Kaji et al. reported that CSE transiently improved balance ability . FurtherVarious exercise methods to improve balance ability such as tiptoe standing, tandem stance, weight shifting, and bracing maneuvers, in addition to traditional CSE, are commonly suggested in the literature ,10,11,12This study was conducted for 3 months at Gachon University. Fifty-eight university students responded to a campus announcement, and 29 met the inclusion criteria and participated in the experiment . The incWe used a crossover design. All 29 participants performed CE and CSE, and to exclude the order effect, a complete counterbalancing method was used; moreover, exercise order was randomized. Measurements were obtained by a physical therapist with a master’s degree and clinical experience of more than 5 years who was blinded to the study.To be able to perform the exercise properly and with the correct movement, the participants performed a familiarization session 1 week prior to the experiment. Before participation, we obtained each individual’s general characteristics, including sex, age, height, and weight. First, surface electromyography (EMG) electrodes were attached to the selected muscles of the participants. The surface EMG and force plate were synchronized. The static balance ability and muscle activation were measured in eye-opened and eye-closed conditions while standing on the force plate. Each exercise (CE and CSE) was performed for 1 min, and static balance ability and muscle activation were measured identically as a baseline after exercises.This exercise consisted of a bracing maneuver, which activates the core muscles, and tiptoe and tandem exercises. The composed exercises were selected as exercises that can improve balance and are easy to perform without space constraints ,12,13,14It was performed by instructing them to “focus on the abdominal muscles and hold their breath after exhalation” and then maintain the abdominal isometric contraction . For theThe participant had a tandem stance posture with the gaze frontward, arms at the pelvis side, and a minimum distance of 5 cm between the front and rear foot. Weight shifting was performed in an anteroposterior direction like a pendulum movement while taking the tandem stance. Weight shifting was performed at a constant rhythm using a smartphone metronome application  adjusted to participants’ preferred speed (1–2 Hz). During weight shifting, the weight shifting range was moved until only the first and second metatarsal heads of both feet touched the ground when moving forward, and only the calcaneus of both feet touched when moving backward. The trunk maintained the bracing maneuver without flexion or extension movement . This exThe participant stood upright with feet shoulder-width apart, staring straight ahead with arms positioned at the sides. Then, the participant was only instructed to raise his heel. During the exercise, the therapist instructed that the bracing maneuver be maintained . This exEach exercise (tandem and tiptoe) was performed for 30 s without resting time between exercises and processed for approximately 1 min in total.As an exercise to stabilize the trunk, CSE consisted of the elbow-toe and hand-heel exercises and was performed as previously described . Each ofTo perform elbow-toe exercise, the knees were extended, the elbows bent at 90 degrees in prone position, and the weight was lifted with the toes and the forearm. The hand was made to hold a fist lightly, the neck was in a neutral position, and the trunk, pelvis, and lower extremity were in a straight line. The thoracic region in which the scapular is located is kept flat by protraction during the plank .To perform the hand-heel exercise, the participant was in a long sitting posture with arms supported and placed heels on a 20 cm-high box. Then, the participant was instructed to raise his hips with elbow extension. The trunk, pelvis, and lower extremity were made to be in a straight line .Each exercise (elbow-toe and hand-heel) was performed for 30 s without resting time between exercises and processed for approximately 1 min in total.To measure postural sway, a force plate  was used, and the signal was processed using MR3.10 Software . During the measurement, the participants took off their shoes and stood straight on the force plate with their arms relaxed comfortably and evenly at their sides. They were instructed to keep their heads as still as possible. Their feet were opened at a 30° angle, and the space between the heels was 9 cm. Participants faced forward and were instructed to stare straight at an X mark (8 cm in width and 6 cm in height) placed at eye level on a wall 1.5 m in front of them . On the 2), center of pressure (COP) path length (mm), COP average velocity (mm/s), length of minor axis (mm), length of major axis (mm), and anterior/posterior foot pressure ratio (A/P ratio). A higher value of the A/P ratio variable means that the center of gravity moved further forward.Recordings were measured for 40 s, and the total measurement of 30 s, which excluded the first and last 5 s, was used for analysis. Additionally, recordings were measured three times each in eye-opened and eye-closed conditions at the baseline and after exercise, and the interval between conditions was set to 10 s. Moreover, as in a previous study, to remove the learning effect, the interval between the measurements at the baseline and after exercise was set to 4 min. To remove the exercise effect, each interval between exercises (CE and CSE) was set to 15 min . The varTo measure muscle activation, 8-channel wireless surface EMG was performed using the Noraxon TELEmyo 2400T  device. The preparation of measurement was completed by removing hairs around the attachment part before placing the electrode and cleaning with alcohol. Pairs of disposable Ag/AgCl surface electrodes were used, and the distance between electrodes was 2 cm. The electrode was attached around the selected muscle toward the participant’s dominance. For raw EMG signal, the frequency of the band-pass filter was set to 20–350 Hz, and the root mean square (RMS) value was set to 100 ms after rectifying and normalizing with maximal voluntary contraction (MVC). MVC was performed in supine , rectus abdominis (RA), tibialis anterior (TA), medial gastrocnemius (GCM)), prone (erector spinae (ES), biceps femoris (BF)), side-lying (gluteus medius (Gmed)), and sitting (rectus femoris (RF)) positions. MVC values were obtained through trunk flexion and left and right twists for IO and RA, trunk extension for ES, dorsiflexion and plantar flexion for TA and GCM, and hip abduction, knee extension and flexion for Gmed, RF, and BF, respectively ,20,21,22The selected muscles were the IO ), RA , ES , Gmed , RF , TA , GCM , and BF . Electrodes were placed as outlined in a previous study ,27,28,29In this study, data are presented as mean and standard deviation and were processed using SPSS 25.0 software . Normality was assessed using the Shapiro–Wilk test. For within-group comparisons, repeated measures analysis of variance, which is a parametric statistical test, was used for variables with normal distributions. On the other hand, the Friedman test, which is a nonparametric statistical test, was used for variables with non-normal distributions. Post-hoc comparison was performed using the Wilcoxon signed-rank test. The statistical significance level was set to 0.05.The characteristics of participants are presented in p < 0.05) (p < 0.05). Result after CSE showed a statistically significant decrease in all variables except the A/P ratio variables in the eye-opened and eye-closed conditions compared with the baseline values (p < 0.05). For comparison between exercises, path length and average velocity variables after CE showed a statistically significant decrease in the eye-opened condition compared with those after CSE (p < 0.05); in the eye-closed condition, the path length variable after CE showed a statistically significant decrease compared with that after CSE (p < 0.05).There was a statistically significant difference in all variables in the eye-opened  and eye- < 0.05) . As a rep < 0.05) (p < 0.05), and the RF, TA, and RF/BF ratio showed a statistically significant decrease compared with baseline values in CSE (p < 0.05). There were no significant differences in all variables among exercises (p > 0.05).In the eye-opened condition, a significant difference was noted in the Gmed, RF, TA, and RF/BF ratio ( < 0.05) . In postp < 0.05) (p < 0.05), and the RF showed a significant decrease compared with those at the baseline in CSE (p < 0.05). There were no significant differences in all variables among exercises (p > 0.05).In the eye-closed condition, a significant difference was noted in the RF and RF/BF ratio ( < 0.05) . In postThis study demonstrated that CE and CSE have a positive impact transiently on healthy male adults’ balance ability and that CE has a more positive impact than CSE. In addition, this study confirmed that both exercises have an impact on muscle activation.Through CE for 1 min, a significant change in score was observed in the balance ability evaluation in the eye-opened  and eye-closed  conditions. In addition, with the eyes open, CE showed a significant decrease in path length and average velocity compared to CSE. Similarly, even with eyes closed, CE showed a significant decrease in path length compared to CSE.The tandem and tiptoe exercises performed in this study generate instability because the base of support is narrow compared to that in a general standing posture, and weight shifting also generates instability . HalvarsDuclos et al. (2004) reported that isometric muscle contraction for 30 s has an impact on posture . They suIn a comparison between CE and CSE, a significant difference in the path length and average velocity seems to affect exercise composition included in CE. Kaji et al. 2010) mentioned that although CSE had a positive effect on static balance, there was no significant difference in postural sway in the anteroposterior direction . In the  mentioneIn the eye-opened and eye-closed conditions for CE, there was a significant decrease in the RF and RF/BF ratio. In order to maintain static balance, the center of gravity (COG) must be located within the base of support (BOS) ,35,36. TIn the eye-opened condition, we identified an increase in Gmed activation after CE. In the case of CSE, base of support is wide, and there is upper limb intervention due to exercise in a supine or prone position. Contrarily, CE is an exercise requiring only the activity of the lower extremity muscles and continuous activity of the Gmed. Therefore, after CE, it seems that the increase in Gmed activation is attributable to the exercise characteristic, and Gmed activation has an impact on balance ability’s improvement.This study has limitations as follows. First, all participants were men; thus, the results cannot be generalized to women. Second, we cannot suggest a specific interpretation of results because there are no studies that provide a reference value for the hamstring/quadriceps ratio. Therefore, a study that can suggest the normal value is needed. Third, the exercise’s long-term effect is uncertain. Future studies should assess the effects of long-term exercise on static standing balance ability, analyze dynamic movement, include athletes who require static standing balance ability as participants, and investigate a more effective exercise program.This study showed that the CE can immediately improve the static balance ability and bring about changes in muscle activity in healthy male adults. It also showed a greater improvement in static balance ability than the CSE, which is commonly used in clinical settings. This result showed the possibility of applying CE to activities of daily living or to those of sports athletes without space constraints. In particular, it is recommended to apply CE as training for healthy persons who enjoy surfing, shooting, and archery as leisure activities or as pre-match exercise for sports players who require static balance skills.


The purpose of this study was to investigate the effectiveness of wideband energy absorbance in diagnosing otosclerosis by comparing the differences in acoustic absorbance between otosclerotic and normal ears. Exactly 90 surgically confirmed otosclerotic ears were included in the test group. The control group consisted of 126 matched normal-hearing subjects. The Titan hearing test platform (Interacoustics) was used for absorbance and acoustic immittance tests. Energy absorbance, measured at tympanometric peak pressure, was analyzed in the range 226–8000 Hz. Differences between normal and otosclerotic ears were analyzed in quarter-octave bands. Wideband absorbance, i.e., absorbance averaged over the 226–2000 Hz band, and resonance frequency were calculated and compared between normal and otosclerotic ears. Significant differences between the absorbance of normal and otosclerotic ears were found, especially at low and middle frequencies. No significant effect of ear side or gender was observed. For average wideband absorbance and resonance frequency, less pronounced  differences were found between normal and otosclerotic ears. Measurement of peak-pressure energy absorbance, averaged over a frequency band around 650 Hz, provides a valid criterion in testing for otosclerosis. The test is highly effective, with a sensitivity and specificity of over 85% and area under receiver operating characteristic curve above 0.9. Average wideband absorbance can also be used, but its effectiveness is lower. Other immittance-related measures are considerably less effective. Otosclerosis manifests in abnormal growth of the ossicular bones and bony fixation of the anterior stapes plate leading to stiffening of the chain. Its typical clinical symptoms are, among other things, progressive conductive or mixed hearing loss, increased or absent acoustic stapedial reflex thresholds (ASRT), and lack of otoacoustic emissions . A comprOtosclerosis affects the mechano-acoustic properties of the middle ear, giving rise to alterations in immittance. The changes are the greatest in the low-frequency region, where middle ear impedance is dominated by stiffness . EffectsAcoustic immittance of the middle ear is measured by reflection of acoustic waves in the ear canal. The key quantities commonly used to characterize middle ear properties are wideband energy reflectance and energy absorbance. The latter expresses the ratio of energy absorbed by the middle ear to the total energy supplied to the ear canal. Energy reflectance is just the complement to the absorbance. Both are convenient measures, taking values between zero and one, and can be easily measured over a wide frequency range by supplying wideband stimuli (clicks or chirps) as test signals ,13. CompMany investigations have confirmed that absorbance in otosclerotic ears is decreased compared to normal-hearing ears, especially in the low-frequency range ,19,20,21The primary aim of the present research was to compare energy absorbance of otosclerotic ears to that of normal-hearing ears. To obtain reliable results, we included a relatively large number of patients with intraoperatively confirmed otosclerosis. The number was greater than those used in similar studies, and we employed a balanced control group. We also checked whether individual characteristics  had an effect on absorbance, and examined whether other immittance-related measures, such as middle ear resonance frequency, could be used as useful criteria for detecting otosclerosis. To complete the picture, we formulated explicit numerical criteria for deciding on the presence of otosclerosis based on each of the immittance-related measures.All subjects gave written informed consent prior to participation in the study. The research procedures were approved by the Ethics Committee of the Institute of Physiology and Pathology of Hearing, Poland, approval No. IFPS:KB/20/2016 of 24 November 2016.All participants underwent comprehensive otologic and audiologic examination which consisted of clinical otoscopy and a battery of audiologic tests, including pure tone and impedance audiometry. In persons in the otosclerosis group, the condition of the middle ear was assessed intraoperatively during stapedotomy surgery.Clinical audiometric assessment of each subject included air-conduction (AC) and bone-conduction (BC) audiometry. Clinical immittance tests consisted of standard 226 Hz tympanometry and ASRT measurements, followed by wideband absorbance measurements.Otologic assessment included examining the ear canal and tympanic membrane by clinical otoscopy in all participants, and in patients from the otosclerosis group a comprehensive intraoperative assessment of the condition of the middle ear. The condition of the tympanic cavity and the shape, possible defects, and mobility of the ossicular chain were documented, concluding with a decision about the type of surgery required.All audiometry tests were carried out in a sound-proof booth. All tests were performed within one day. In persons from the test group, it was a part of a pre-operative examination of them as a candidate for ear surgery.Tonal audiometry was performed with a Madsen Astera clinical audiometer (Interacoustics). Hearing thresholds in air were determined at 0.125, 0.25, 0.5, 1, 2, 4, and 8 kHz, and for BC at frequencies of 0.25, 1, 2, and 4 kHz. For all ears the air–bone gap was calculated.For impedance audiometry testing a Zodiac 901 Middle Ear Analyzer was used. Clinical immittance tests contained standard 226 Hz tympanometry and ASRT measurement using tone activators at 0.5, 1, 2, and 4 kHz in the ipsilateral mode, and the same tone frequencies in the contralateral mode. Activator level was limited to 110 dB SPL for tonal activators. The absence of an ASRT was marked as 100 dB SPL.For wideband absorbance measurements we used the software package (IMP440/WBT440 version v.3.2) for impedance and wideband tympanometry supplied with the Titan (Interacoustics) system, in the measurement set-up shown in The instruments were periodically calibrated in the manufacturer’s laboratories. A set of standard acoustic cavities was used to calibrate the source in order to set operating parameters. Additionally, before each series of tests, the stability and repeatability of the instruments’ performance was verified by measuring absorbance of an ear simulator (Brüel and Kjær head and torso simulator BK4128C). A set of absorbance–frequency characteristics of the ear simulator, measured with the seven instruments used in this study, is presented in Measured absorbance data were extracted from the patient’s file (xml format) and frequency, pressure, and absorbance characteristics were stored in a numerical file. From the same source of data we extracted resonance frequency (FR), static compliance (SC) at 226 Hz, and other tympanometric parameters.The Titan IMP440 instrument makes it possible to measure, among other things, the so-called wide-band absorbance, averaged over a certain frequency band. In the default test protocol, these bands are either 375–2000 Hz (for adults), or 800–2000 Hz (for testing children under 6 months of age). The bands, proposed by researchers who originally developed the method , are oftIt is known from other studies  that thethe probe placed in the first ear (randomly chosen);the probe removed and placed in the contralateral ear;the probe returned to the first ear, and the test repeated;the test repeated in the ipsilateral ear.It is known that one of the main sources of absorbance measurement errors is improper insertion of the probe in the ear canal  ,25,26,27We expected that satisfactory repeatability of results would be evidence that the probe was correctly placed in both sets of measurements. In such cases, an average of the two absorbance tests was calculated and stored as the final result. Repeatability of the absorbance–frequency characteristics was assessed by calculating the average discrepancy between the results, defined as the square root of variance between the tests averaged over the whole frequency range . We stipulated that this error must not exceed 0.05. Excessive errors were rejected, or the tests were repeated in a following session.The data collected from the tests was used to determine the following: (i) the characteristics of absorbance vs. frequency, measured at TPP; (ii) SC at 226 Hz; and (iii) FR.For statistical analyses, the interval 226 to 8000 Hz was divided into 21 quarter-octave bands with center frequencies of 241, 288, 343, 408, 485, 581, 696, 828, 985, 1172, 1393, 1657, 1971, 2344, 2787, 3315, 3942, 4688, 5575, 6630, and 7660 Hz. For each band the average absorbance was calculated, denoted AA241, AA288, AA343, and so on. We also calculated the average absorbance over the 226–2000 Hz interval, denoted AA2000, similar to that which is displayed in the Titan system as the peak value of the “wideband tympanogram”.t-tests were used to compare absorbance, SC, and FR between the groups. Fisher’s exact test was also used to compare gender difference between otosclerotic and control groups.When comparing absorbances between the otosclerosis and control groups, we applied a mixed-model analysis of variance (ANOVA). In this model, gender  and group (norm and otosclerosis) were the between-subject factors, and ear (left and right) and frequency were the within-subject factors. Independent samples To assess the accuracy of classifying ears as normal or impaired, we determined the sensitivity and specificity of tests based on different sets of criteria. The quantities considered were average absorbance in various frequency bands, SC and FR.Receiver operating characteristic (ROC) and area under the ROC curve (AUC) were used to gauge the efficiency of the methods. An ROC curve plots the relative proportion of hits (sensitivity) against the number of false alarms (1—specificity). Sensitivity (SEN) is the likelihood of identifying an ear as impaired when hearing loss  is present; specificity (SPE) is the odds of identifying a normal-hearing ear as normal; and efficiency is the proportion of ears that are correctly identified. AUC ranges from 0.5 for a test with no diagnostic power to 1.0 for a test with perfect diagnostic ability.For analyses we used Statistica v. 12  and Matlab with Statistical Toolbox R2019b .Two groups of subjects were selected for this study: otosclerotic patients (the test group) and a cohort of normal-hearing persons (the control group). Later these groups are referred to as Otosclerotic and Normal.The Otosclerotic group consisted of patients admitted to the Oto-Rhino-Laryngosurgery Clinic of the Institute as candidates for stapedotomy surgery. All patients were thoroughly examined before surgery, and otosclerosis was diagnosed based on clinical criteria: progressive conductive hearing loss, absence or abnormally high ASRT, absent otoacoustic emissions, and the results of otoscopic examination . The presence of otosclerosis was then confirmed intraoperatively during middle ear surgery . All theFrom this group of patients, we excluded cases of (i) reoperation, (ii) multiple pathologies, and (iii) severe malformations of the middle ear.The Normal group was recruited from volunteers. All candidates were comprehensively examined to confirm lack of hearing impairment. Inclusion criteria were: normal air-conduction hearing threshold level (≤20 dB HL in the 125–8000 Hz range), air-bone gap ≤10 dB from 0.5 to 4 kHz, ASRT <100 dB SPL, ear canal and tympanic membrane having no pathological changes or malformations as confirmed by otoscopic examination, and normal tympanometric characteristics at 226 Hz ,30,31 (iAges and genders of both groups are listed in t-test p < 0.0001). Also, there was a significant difference in age between men and women in the Normal group (p = 0.006); however, there was no such difference in the Otosclerotic group (p = 0.39). There was almost the same number of left and right ears in both groups, and a similar proportion of men to women (38/88 and 25/65 respectively). A χ2-test showed no significant difference between them (p = 0.704).As one might expect, the average age (both genders) in the Otosclerotic group was significantly greater than in the Normal group  as within-subject factors, group  and gender  as between-subject factor, and frequency as repeated-measure factor (FREQ). F = 119.2, p < 0.001) and FREQ (frequency) . Significant interaction effects were observed between FREQ and Group , FREQ and Gender , and Group and Gender . The remaining effects and interactions were below the level of significance. The complete results of ANOVA are listed in The only significant main effects were for Group  of a test that might be applied for detecting otosclerosis based on absorbance as the test criterion. AUC is a convenient and widely used measure for assessing the effectiveness of a test ,33. AUC p < 0.05) in almost all considered bands, except for AA4688, AA5575, and AA6630. The greatest differences  appear in some low and medium frequency bands (AA408–AA828), and in AA2000 . In all these cases, AUC takes maximum values of ≥0.86. It therefore seems reasonable to use absorbances from these bands as a criterion for otosclerosis.As shown in According to the above results, the most useful measure would be absorbance averaged in the frequency interval encompassing the range of maximum AUC, i.e., the 545–749 Hz band. Average absorbance in this band will henceforth be denoted AA650. Other immittance-related measures that might be taken into account as test criteria are AA2000, SC, and FR.The histograms in SEN, SPE) determined experimentally in that sample might differ from the “true” values determined in the entire population [n1p1 (= n1⋅SEN = TP) and n2p2 (= n2⋅SPE = TN) have independent binomial distributions ipin ~ B, where i = 1, 2 and n1, n2 are the numbers of impaired and normal ears in the population sample, respectively; p1, p2 stand for the sensitivity and specificity of the test; and TP, TN are the numbers of true positive and true negative test results. We calculated  confidence intervals for TP and TN directly from the appropriate binomial distribution functions. As Based on the distributions in pulation . To assepulation  that botAs these figures show, the courses of the sensitivity and specificity functions differ depending on the applied test criterion. The best performance of test is achieved at the intersection point of sensitivity and specificity values. We may estimate these values as approximately 0.86 for AA650, 0.8 for AA2000, 0.75 for SC, and 0.55 for FR.–1 (1—SPE) and Φ–1 (SEN) [–1 (SEN) .For normally-distributed test results of true-positives and false-positives, a graph of SEN vs. (1–SPE) plotted in nonlinear coordinates of units of inverse normal distribution would take the form of a straight line . If its We have also examined relationships between the applied immittance-related measures. p < 0.05) with correlation coefficients r in most cases above 0.5, which denotes a large effect size.As one might expect, correlations between all these variables are significant  and rely on typical test protocols to measure immittance-related quantities. Unlike some other similar studies ,36,37,38It is known that experimentally determined absorbance (as well as other similar measures) exhibit significant variability, both in normal hearing subjects  and in pπ/2 phase shift) of the middle ear admittance [Assessment of the effectiveness of various tests for otosclerosis based on immittance measures has been the subject of a number of studies ,40,41,42mittance ,14; and mittance . It has mittance ,14,30.In order to achieve the above-mentioned aim of the research, we decided to eliminate sources of errors as much as possible and look for alternative measures that could be used as test criteria. The accuracy of the instrumentation was carefully checked, instrument readings were verified with the use of an admittance standard , and we ensured that testers had undergone practical training on a group of healthy volunteers before they started examining patients. To limit errors resulting from improper insertion of the probe in the ear canal, we instructed the testers to repeat the test twice, each time re-inserting the probe from ipsi- to contralateral ear. Absorbance from two insertions in the same ear were compared and a consistency measure was calculated based on the average variance between two tests. In this way, we were able to obtain reliable results in the majority of examined ears. Only in a small number of cases (less than 5%) did we get outliers, and these were identified as being due to problems with non-cooperative patients, additional ear problems such as fluid in the tympanic cavity, cholesteatoma, etc., and these results were neglected.Personal characteristics of both examined groups  were compared statistically. The greater number of women in the Otosclerotic group results from the greater prevalence of otosclerosis in females . There wIn order to analyze the impact of otosclerosis on frequency characteristics, as well as the effect of personal characteristics in both groups, we applied a repeated-measures ANOVA model, taking “group” and “gender” as inter-personal factors, “ear” as the intra-personal factor, and frequency as the repeated-measure factor . The analysis showed very significant effects of “group” and frequency, which is already well understood see . There wOur results show the greatest differences in absorbance between the groups in the low and medium frequency range, up to 2000 Hz. It therefore makes sense to investigate this region and examine whether there are any measures that might be used as a criterion for otosclerosis. Because the distributions of absorbance values, in both groups and in practically all frequency bands, do not fulfill the criteria of normality, we applied a non-parametric Mann–Whitney U-test to compare groups. The test confirmed significant differences at almost all frequencies (except in the 4.7–6.6 kHz range); moreover, it allowed specific AUC values of tests for otosclerosis to be calculated. The larger AUCs, above 0.9, appear in the frequency range of approximately 550 to 700 Hz, so we decided to take the absorbance averaged over this band, denoted AA650, as a test criterion for further investigation. The other quantities considered were absorbance averaged over the 226–2000 Hz band , FR, and SC.Based on the experimentally determined statistical distributions of test results for the Normal and Otosclerotic groups, we calculated SEN and SPE characteristics of tests based on each criterion. As seen in Application of SC to tests for otosclerosis needs to be specifically addressed. Theoretically, based on the distributions of the examined groups, one can expect a sensitivity and specificity of approximately 0.75 when SC is chosen as the test criterion (for a normative SC ≈ 0.46 mL). However, it needs to be remembered that, following what is generally assumed in audiology, the criterion for inclusion to the norm group required SC should be ≥0.3 mL ,30. TherFR seems to be the measure least suitable for an otosclerosis test. In theory, otosclerosis should cause a significant increase in FR due to an increase in stiffness of the ossicular chain. However, the observed variability of FR is large, again consistent with observations of others . AlthougAlthough the sensitivity and specificity of an otosclerosis test based on some absorbance-related measure may be relatively high (above 85%), one may still need to improve it. Theoretically, it is possible to apply a test battery consisting of multiple tests based on different absorbance-related measures. However, this is probably not practical, because all the above-described measures  are strongly correlated . This meIt is generally agreed that testing energy absorbance of the middle ear can provide useful information about its status and facilitate detection of ear pathology. However, the applicability and reliability of these methods is still controversial. Our investigation focused on examining possible ways of using immittance-related tests to diagnose otosclerosis. Considering the number of otosclerotic patients who qualify for stapedotomy (and other reconstructive surgeries) in our clinic, any reliable objective diagnostic method which permitted early detection of otosclerotic changes would be particularly useful.The investigation here has shown that a reasonably accurate method of testing for the presence of otosclerosis can be created based on energy absorbance measurements obtained with a standard, commercially available instrument.The most suitable quantity for this purpose is the energy absorbance measured at tympanometric peak pressure for low frequencies, a region where otosclerotic changes readily affect mechanoacoustic properties of the middle ear. We found that the optimal measure to use as a test criterion is the average absorbance over the band of about 550 to 750 Hz. Our results demonstrate that, using this measure, one can achieve high sensitivity and specificity (above 85%) and high accuracy, described with an AUC of greater than 0.9. The “wideband tympanogram” measure, which is implemented in the current commercial WBT system, can also be used, but it is less effective. There seems to be very limited usefulness of other immittance-related measures such as SC and FR.An unexpected finding in this study was the effect of gender in the group of otosclerotic patients, which appeared in the higher frequency range. This suggests that gender might be considered as a variable in future studies on immittance measures in otosclerosis.Our experience, and the opinions of other researchers, is that reasonably accurate absorbance measurements can be achieved with commercial systems. However, it needs careful verification of system functioning, frequent and precise calibration (more precise than provided in standard maintenance), and measures to limit random errors .Further investigations are needed to refine the method. Among important problems, one could evaluate postoperative changes in middle ear absorbance and relate them to audiological effects. Another interesting field of investigation would be patients undergoing other types of ear surgery, including cochlear and middle ear implants.


Our goal was to compare the operative and postoperative effects of del Nido cardioplegia (DN group) and blood cardioplegia (BC group) performed in cardiac surgery.A total of 83 patients were included, separated into DN group and BC group. The operative and postoperative effects of the two groups were compared for the first 24 hours until extubation. The operative and postoperative complete blood count (CBC), biochemical values and clinical parameters were compared.P=0.003) during the operation. The amount of cardioplegia in DN group were lower than that in BC group (P=0.001). The pump outflow and postoperative lactate level of DN group were lower than those of BC group , as well as the amounts of NaHCO3 (P=0.006) and KCl (P=0.001) used during the operation. The same occurred with the first monocytes (Mo) and mean corpuscular volume (MCV) levels in the postoperative intensive care unit . However, the first glucose level and the eosinophil (Eo) level were higher in DN group .The first control activated clotting time (ACT) levels in DN group patients were lower (3 and KCl in DN group were lower. In postoperative evaluation, measured level of lactate, Mo and MCV in DN group were all lower; their glucose and Eo levels were higher.In the operative evaluation, the amount of cardioplegia, the first ACT levels, the pump outflow lactate level and the amounts of NaHCO Cardioplegia, as well as local and systemic hypothermia, have been used in cardiac surgery for many years for myocardial protection,4. The causes of myocardial damage are global myocardial ischemia (aortic cross-clamp) and especially reperfusion. Reperfusion following the ischemic period, although a definite need to maintain the viability of ischemic tissue, can itself cause serious or even potentially lethal damage to tissue that otherwise could have survived. Cardioplegia, which is an elective and chemical cardiac arrest technique, was first applied as potassium cardioplegia in 1955 in cardiovascular surgery by Melrose. Researchers at the University of Pittsburgh  developed a new formulation for myocardial protection in the early 1990s. This team, led by Pedro del Nido, Hung Cao-Danh, K. Eric Sommers, and Akihiko Ohkado, patented this solution as a result. Changes have been made to the original solution and, in the literature, its use in clinical practice is referred to as del Nido cardioplegia.In cardiac surgery, the myocardial injury sustained during the operation is the most important cause of mortality and morbidity. The application of cardiopulmonary bypass (CPB) and elective cardiac arrest provided surgeons with the ability to operate in a blood-free environment and the precious time necessary for meticulous work. Ensuring adequate myocardial protection during the surgery, as well as before and after the operation, is the most significant factor of successOur goal in this study was to compare the effects of del Nido cardioplegia with those of blood cardioplegia, both during the operation and in the first 24 postoperative hours until extubation, in patients undergoing CPB with the mechanical ventilator turned off throughout the pumping period.Approval for this study was granted (#2018/12-06) on July 13, 2018, by the Clinical Research Ethics Committee of the Faculty of Medicine, Kahramanmaras Sutcu Imam University. All patients included in the study had on-pump CPB between May 1, 2017, and August 15, 2018. Patient files and hospital information management system (HIMS) records were retrospectively reviewed and, of 150 cases operated on between these dates, 83 were included in the study.3, 84 mg/mL, ampoule), potassium chloride , magnesium sulphate , Calcium Picken , furosemide , and 20% mannitol (100 ml) used during CPB. Complete blood count (CBC) and biochemical and albumin levels, the amount of urine produced prior to extubation and time of extubation were all recorded in the intensive care unit (ICU) for the first 24 hours after surgery. The use of operative and postoperative inotropic agents and the development of postoperative atrial fibrillation in the ICU were investigated.Patients were divided into two groups: those who received del Nido cardioplegia  and those who received blood cardioplegia . Components of the solution used in el Nido and blood cardioplegia was shown in 2 0.5 or 50%, tidal volume 7 to 10 mL/kg, respiratory rate 12, inspiration/expiration rate 1/2, flow of 2 L/min.Surgery was performed by two separate surgical teams at the cardiovascular surgery clinic. In all cases, antegrade cardioplegia was used and CPB with mild systemic hypothermia (30 to 34°C). Anesthesia was induced with 0.01 mg/kg of midazolam, 5 to 10 µg/kg of fentanyl, and 0.1 mg/kg of pancuronium. Pancuronium was administered continuously every half hour. Left radial artery cannulation was performed, followed by central venous catheterization. Sevoflurane 1-2 minimum alveolar concentration (MAC) was used in the operation. The mechanical ventilator settings were: FiOP<0.05. Data were evaluated with IBM SPSS 22 software.Data were analyzed by the Shapiro-Wilk test, while the independent samples t-test was used to examine the differences between the BC and DN cardioplegia groups. Statistical parameters were expressed as mean±SEM. We examined the distribution of categorical variables according to groups using the chi-square test. In categorical variables, the statistical parameters were expressed as percentages (%) and frequencies (n). Statistical significance was accepted as The mean age of the patients in DN group was 54.95 years and those in BC group was 59.83 years. There were 22 male patients (51.16%) in DN group and 23 (57.50%) in BC group .Coronary artery bypass grafting (CABG) patients comprised 41.8% of the DN group members and 57.5% of the BC group. Among the comorbidities observed in patients, hypertension was quite high .P=0.003). The amounts of cardioplegia used were 955.81±32.78 mL in DN group and 1667.74±84.03 mL in BC group, and this difference was statistically significant (P=0.001). The pump outflow lactate level of the DN group was also lower by a statistically significant amount (P=0.005). The amount of NaHCO3 and KCl used during the surgeries were also lower for DN group patients, with statistically significant differences  (P=0.018). From the postoperative CBC tests, the first monocytes (Mo) and mean corpuscular volume (MCV) levels in the DN group were both low when compared to those in the BC group. The differences were statistically significant . In the postoperative ICU, the first measured glucose level and eosinophil  level were higher in DN group, and this was statistically significant  (P=0.013). There was no difference between the groups in terms of postoperative atrial fibrillation development (P=0.692)  . In the ctively) . The numP=0.692) .3 used, and the amounts of KCl in DN group were all lower. In the postoperative ICU evaluation, first lactate level, Mo, and MCV levels in DN group were all lower and the first glucose levels and Eo were higher than those of BC group.In the operative evaluation of our study, the amount of cardioplegia (mL), the first measured ACT values, the outflow lactate level, the amounts of NaHCO,11. In another study, comparing the use of del Nido with that of St. Thomas’ cardioplegic solution, it was shown that del Nido cardioplegia resulted in shorter cross-clamp and CPB times, less cardioplegia used, and better myocardial protection in terms of left ventricular ejection fraction (LVEF). The CPB times in our study were shorter for BC group than for DN group, but this difference was not statistically significant. In a separate study, cardiac ischemia and total CPB times were shorter in DN group than in BC group. This difference is probably attributable to the normothermic strategy used in the DN group and the feasibility of the single-dose infusion. Del Nido cardioplegia has been shown to be safe in the reoperative aortic valve and post-infarction CABG surgeries. It has also been reported not only to reduce operative time but also to result in myocardial protection and cost-effectiveness in isolated valve surgeries. In the postoperative ICU evaluation of our study, glucose levels in DN group were higher than those of BC group. A different study reported that the myocardial protection and clinical results provided by del Nido cardioplegia in adult isolated CABG patients is equal to that provided by blood cardioplegia, while the CPB glucose levels of del Nido are lower than those of blood cardioplegia. There was no difference in the operative blood glucose level in our study.In studies similar to ours, a smaller amount of cardioplegia (mL) were used in DN group. Myocardial injury measurements, such as troponin T levels, postoperative LVEF, and postoperative inotropic/vasopressor support, were similar to those in patients who received Buckberg solution. The crystalloid component of the del Nido solution is not glucose-based (unlike the Buckberg solution); patients who received the del Nido solution had lower blood sugar levels and needed less insulin in the postoperative period. In our study, the number of patients using noradrenaline during the operation was higher in the DN group. But there was no difference between groups in terms of postoperative inotropic use. In addition, there was no difference between the two groups in terms of the development of postoperative atrial fibrillation. Del Nido cardioplegia has been shown to be associated with reduced spontaneous activity and myocardial injury, and with better functional recovery during arrest in isolated older hearts, when compared to blood cardioplegia. In one study, del Nido cardioplegia solution had the potential to provide superior myocardial protection in elderly hearts by preventing electromechanical activity during cardioplegic arrest and Ca2+-induced hypercontraction during early reperfusion. Studies have shown that the sustained release of lactate or higher lactate levels in the reperfusion period after CABG is not satisfactory for myocardial protection and the improvement of aerobic metabolism decreases perioperatively.Del Nido solution is safe for use in adult primary isolated aortic and mitral valve operations. Some surgical approaches have the advantage of reducing surgical time, cost, postoperative insulin need, surgical downtime, and also give a more positive outcome with regard to blood glucose levels3 and KCL used were higher in DN group, the low amounts of NaHCO3 and KCL used throughout the operation were considered normal.In our study, the pump outflow and postoperative lactate levels in DN group were lower than in BC group. The low operative and postoperative lactate levels in DN group, and its smaller amount of cardioplegia, were found to be advantageous in terms of myocardial protection and clinical follow-up. Since the amounts of NaHCOIn the postoperative ICU evaluation, the lower the first measured Mo and MCV values, the higher the first glucose level, which can be seen as a disadvantage of del Nido cardioplegia. It is our belief that our study will constitute an example in the literature and lead to new studies. The limitations of our study are its retrospective nature and the diversity of cases.In our study, the operative and postoperative effects in DN group and BC group were investigated. In the operative evaluation, the cardioplegic amount (mL) used and the pump outflow lactate level in DN group were lower. This was an advantage in terms of myocardial protection and clinical follow-up. In the postoperative evaluation in the ICU, the first measured level of lactate, Mo, and MCV in DN group were all lower, while the first glucose level was higher. The low postoperative lactate level was considered an advantage of del Nido cardioplegia, whereas the lower the Mo and MCV values, the high first glucose level, which was seen as a disadvantage. These results will bring different perspectives on the use of del Nido and blood cardioplegia and will pave the way for new studies.