Pancreatic cancer (PC) is among the most lethal cancers. The lack of effective tools for early detection results in late tumor detection and, consequently, high mortality rate. Precision oncology aims to develop targeted individual treatments based on advanced computational approaches of omics data. Biomarkers, such as global alteration of cytosine (CpG) methylation, can be pivotal for these objectives. In this study, we performed DNA methylation profiling of pancreatic cancer patients using circulating cell‐free DNA (cfDNA) and artificial intelligence (AI) including Deep Learning (DL) for minimally invasive detection to elucidate the epigenetic pathogenesis of PC.The Illumina Infinium HD Assay was used for genome‐wide DNA methylation profiling of cfDNA in treatment‐naïve patients. Six AI algorithms were used to determine PC detection accuracy based on cytosine (CpG) methylation markers. Additional strategies for minimizing overfitting were employed. The molecular pathogenesis was interrogated using enrichment analysis.q‐value < 0.05; Bonferroni correction) in PC versus controls. Highly accurate PC detection was achieved with all 6 AI platforms (Area under the receiver operator characteristics curve [0.90–1.00]). For example, DL achieved AUC (95% CI): 1.00 (0.95–1.00), with a sensitivity and specificity of 100%. A separate modeling approach based on logistic regression‐based yielded an AUC (95% CI) 1.0 (1.0–1.0) with a sensitivity and specificity of 100% for PC detection. The top four biological pathways that were epigenetically altered in PC and are known to be linked with cancer are discussed.In total, we identified 4556 significantly differentially methylated CpGs (Using a minimally invasive approach, AI, and epigenetic analysis of circulating cfDNA, high predictive accuracy for PC was achieved. From a clinical perspective, our findings suggest that that early detection leading to improved overall survival may be achievable in the future. AI is a branch of computer science where machines can synthesize data presented to them, learn patterns therefrom, and identify these patterns and associations in new datasets. An exciting application of AI is the ability to identify previously unrecognized, defining features in a dataset and accurately classify or distinguish groups. In the biological sciences, AI can now be coupled with genomics for highly accurate disease detection and to elucidate the mechanisms of complex disorders including cancer.2The Institutional Review Board of Beaumont Health, Royal Oak, MI, approved the study protocol (IRB#2018‐306). Participants provided written consent. Blood was obtained from seven treatment‐naïve PC patients and with 14 controls who had no diagnosis or suspicion of cancer. All the PC cases had histological confirmation of the diagnosis, and none received radiation, chemotherapy, or surgical therapy prior to sample collection. The specimens were collected in Streck Cell‐Free DNA BCT® tubes,2.1Raw iDAT files of Illumina EPIC array data were processed using the R package (v 4.1.1). The package's “minfi” and “noob” normalization method was used for data normalization. Outlier detection was performed, and two control samples were considered as outliers2.2A graphical enrichment analysis tool “ShinyGO 0.77” was used to perform the gene enrichment analysis. Enrichment of pathways was performed using the previously described approaches.2.3https://www.geneimprint.com/site/genes‐by‐species) to help elucidate the role of imprinted genes in PC pathogenesis.We performed a search using “geneimprint” database  and logistic regression models were performed using the R package through MetaboAnalyst (v 5.0).3There were a total of seven treatment‐naïve PC cases and 12 unaffected controls analyzed after elimination of two outliers. The detected outliers are pictorially represented in Figure p‐value < 0.05) differentially methylated in PC versus controls. A total of 2805 hypomethylated and 1751 hypermethylated CpG markers were identified in the study with the FDR‐adjusted p‐value < 0.05. The CpGs were significantly more likely to be hypo‐ rather than hypermethylated  in PC  of 1 (0.95–1) with sensitivity and specificity of 100% for both intragenic and separate intergenic CpG markers. The DL intragenic CpG markers using 10 predictive variables  were: cg16984992, cg16590012, cgg16550438, cg07240877, cg14870958, cg01291513, cg13151361, cg21242417, cg09518293, and cg00019091 Table  and S9.3.3The PLS‐DA plot showed good separation between the PC and control groups Figure . The R2 The literature indicates that AI is superior to regression analysis for group discrimination.4The integration of systems biology including epigenomics, genomics, and transcriptomics has advanced our understanding of the mechanisms of pancreatic cancer. Further, targeted therapy based on these molecular alterations appears to be associated with an improved prognosis.The late presentation and diagnosis of PC remain a fundamental challenge to improving survival statistics.NPTX2 gene methylation achieved sensitivity of 42% and specificity of 98% for PC detection. However, the need to collect pancreatic juice, which is not easily accessible, would preclude the use of this approach in the routine screening of at‐risk individuals. As noted above, our parsimonious algorithm based on five markers and using DL achieved an AUC (95% CI) = 1 (0.95–1) with a sensitivity and specificity of 100% when either intragenic or intergenic CpGs by themselves were used , kininogen (KNG1), ARVCF, and Apolipoprotein E (APOE) have also been identified as carriers of the CA19‐9 antigen.MUC4, MUC6, and MUC16. Our work helps to lay the groundwork for this effort in identifying epigenetically dysregulated loci and molecular pathways associated with pancreatic cancer.While epigenomic therapeutics offer promise in pancreatic cancer therapy,Our study is not without limitations. These include the relatively small sample size. Multiple AI strategies, including choice of platforms as detailed in Data The ultimate objective of precision oncology is the development of targeted, patient‐specific treatments. The fundamental challenge with current cancer therapy is the clonality and heterogenetic of tumor cells resulting in initial suppression, but subsequent resurgence of resistant clones leading to drug resistance, and clinical recurrence. There is now good evidence that epigenetic modification plays a critical role in tumor cell heterogeneity.In summary, using a minimally invasive approach based on epigenome‐wide analysis of circulating cfDNA, consistently high PC prediction was achieved. Further, our analysis found that the genes and molecular pathways that were epigenetically altered are known or suspected to be involved in various aspects of cancer biology giving further biological plausibility to our findings. Larger studies to validate our findings are clearly indicated.Ray O. Bahado‐Singh: Conceptualization ; data curation ; funding acquisition (lead); investigation ; project administration ; resources ; supervision ; writing – original draft . Onur Turkoglu: Data curation ; formal analysis ; software ; writing – review and editing . Buket Aydas: Data curation ; formal analysis ; methodology ; software ; writing – review and editing . Sangeetha Vishweswaraiah: Conceptualization ; data curation ; investigation ; methodology ; project administration ; software ; supervision ; validation ; visualization ; writing – original draft .These data have partly contributed to a provisional patent application by the home institution, Beaumont Research Institute, for cancer detection.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Figure S4.Click here for additional data file.Figure S5.Click here for additional data file.Figure S6.Click here for additional data file.Figure S7.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.Table S3.Click here for additional data file.Tables S4–S9.Click here for additional data file.Data S1.Click here for additional data file.


To investigate the relationship between the incidence of atrial fibrillation (AF) recurrence and the levels of the systemic immune‐inflammatory index  in patients with AF and diabetes mellitus (DM) undergoing after radiofrequency catheter ablation (RFCA).Preoperative SII levels were determined in AF patients with DM undergoing RFCA. Restricted cubic splines were used to determine the correlation between SII and the risk of AF recurrence. Multivariate‐adjusted logistic regression models were constructed to determine the relationship between SII levels and AF recurrence. The predictive value of the clinical model and combined with the SII index was estimated by the area under the receiver‑operating characteristic curve, net reclassification improvement (NRI), and integrated discrimination improvement (IDI).9/L, there was a positive correlation with the incidence of AF recurrence. In addition, adding the SII to the predictive model for AF recurrence after RFCA in patients with DM and AF could contribute to an increase in C‐statistics . After SII was incorporated into the clinical model, the comprehensive discrimination and net reclassification tended to improve .A total of 204 patients with AF and DM who underwent RFCA in our hospital were included. Seventy‐seven patients had AF recurred during a mean follow‐up of 20 months. Restricted cubic spline analysis showed that when SII ≥ 444.77 × 10SII was independently and positively associated with recurrence after the first catheter ablation in patients with DM and AF. Atrial fibrillation (AF) is the most common cardiac arrhythmia and radiofrequency catheter ablation (RFCA) has been widely used in treatment for AF, but the high rate of recurrence after ablation remains a significant clinical problem. Systemic immune‐inflammatory index is an independent risk predictor of recurrence after radiofrequency ablation in patients with AF and diabetes mellitus (DM). Based on this study, clinicians may provide preventive treatment for patients with AF and DM who will be under RFCA. Therefore, this study evaluates the relationship between SII and the outcome after RFCA in a cohort of patients with coincidence of AF and DM.22.1The flow diagram of our study was shown in Figure 2.22DS2‐VASc score, hypertension, and coronary artery disease were all collected from the electronic medical recording system by trained physicians who were blinded to the aim of the study. In addition, blood markers were recorded including fasting plasma glucose (FBG), glycosylated hemoglobin (HbA1c), serum creatinine, serum uric acid, estimated glomerular filtration rate (eGFR), urea, cystatin C, serum lipid profiles including TG, total cholesterol, low‐density lipoprotein‐C, and high‐density lipoprotein‐C, counts of white blood cells, neutrophils, lymphocytes, platelet, hemoglobin, high sensitivity‐C reactive protein (hs‐CRP). Cardiac ultrasound, 12‐lead electrocardiogram (ECG), and 24‐h ECG were obtained for analysis.Patient demographics and clinical characteristics, including age, sex, body mass index (BMI), smoking history, type of diabetes, type of AF, duration of AF, CHA2). The diagnosis of DM was confirmed by a history of DM, active treatment with antidiabetic medication, or the typical symptoms of DM with FBG ≥7 mmol/L and/or random blood glucose ≥11.1 mmol/L.The SII was determined with the following formula: neutrophil × platelet/lymphocyte, the neutrophils to lymphocytes (NLR) index was determined with the following formula: neutrophil/lymphocyte and BMI was calculated as weight (kg)/height squared , left ventricular ejection fraction (LVEF); and model 4, which is the fully adjusted model, was adjusted for variables in model 3 plus HbA1c, eGFR, hs‐CRP. Furthermore, to evaluate the predictive value of the SII for AF recurrence, the area under the curve (AUC) and the optimal cut‐off value were assessed through receiver operating characteristic (ROC) curve analysis. Moreover, the AUC of NLR was calculated and compared with SII. Meanwhile, to evaluate whether introducing the SII into the model of established risk factors could improve the predictive value, the C‐statistic was calculated and compared by De‐Long's test. Additionally, net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were also calculated to further evaluate the incremental predictive value of the SII index. All statistical analyses were performed using SPSS version 26.0 (SPSS Inc.), and the statistical package R, Version 4.0.3 (https://cran.r-project.org) were prepared. All tests were two‐tailed, and p < .05 was considered statistically significant.Categorical variables were expressed as counts and percentages (%), while continuous variables were expressed as mean standard deviation or median and interquartile range. To identify determinants of recurrence in AF patients with DM after RFCA, a univariate logistic regression analysis was performed. The baseline variables were selected and included in the multivariable logistic regression analysis model if they showed 33.1p < .05).A total of 204 patients who underwent RFCA at a median follow‐up time of 20 months after successful RFCA were enrolled. As shown in Table 3.2p < .001). Additionally, it is noteworthy that the recurrent group also had a significantly higher SII index than the non‐recurrent group  were independent risk factors predicting the AF recurrence after RFCA , and the association persisted in the four models. In the fully adjusted model 4, the risk of recurrent AF was higher in patients who had SII levels above 444.77 than those with levels below the cut‐off   Table .3.3p < .001). The optimal cut‐off value was 444.77 . Moreover, the AUC of NLR is 0.648 . The optimal cut‐off value was 2.55 . Meanwhile, the C‐statistic obtained from the model of established risk factors, which consisted of AF type, LAD, and HbA1c, was 0.749 . Furthermore, Table p = .034), NRI , and IDI .As presented in Figure 4In this comprehensive analysis, we investigate the relationship between inflammation assessed by the SII and AF recurrence in patients with DM undergoing RFCA. The SII was significantly associated with cardiometabolic risk factors. And patients with a higher SII were more likely to have a high risk of AF recurrence after successful RFCA. Besides, SII has a better predictive value than NLR. The SII, either as a continuous or categorical variable, was independently associated with an increased risk of AF recurrence in the fully adjusted model. Besides, LAD, type of AF, and HbA1c were found to be independent risk factors for AF recurrence in patients with DM combined with AF, respectively. In addition, taking the SII into consideration may have clinical significance in the majorization of the early risk stratification of AF recurrence in patients with DM undergoing RFCA.AF is the most common cardiac arrhythmia worldwideHigher levels of inflammation were found in DM than those in non‐diabetic patients.The SII can reflect the inflammatory and immune state of the body, which has the characteristics of cheap price and ease of detection in the clinic.There are several limitations to this study. First, this was a single‐center retrospective study, and the inherent limitations of such studies inevitably affect patient selection and create selection bias. Second, we calculated SII only once at admission and did not monitor changes in these inflammatory biomarkers during the study period. Also, patients with asymptomatic AF maybe ignored without longer periods of ambulatory monitoring. Third, the cases in this study were from a single source, and the sample size is small, the conclusion needs to be further explored by multicenter and expanded sample size.Our present work showed that the SII was an independent predictor of AF recurrence in patients with AF and DM. Based on our findings, early evaluation of AF recurrence risk and interventions may be important for the prevention of AF recurrence in the future.5Consistent with previous studies, our present study suggested that introducing the SII into a model of established risk factors could improve our ability to identify patients at risk for AF recurrence. Although its incremental predictive value for AF recurrence was limited, considering the large and increasing number of patients with AF and DM admitted for RFCA every year, it still seems to be clinically important to perform assessments of the risk of AF recurrence combined with established risk factors.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supplementary Figure 1: Forest plot of the multivariable logistic regression analysis model in patients with DM exploring the association between SII and AF recurrence after RFCA. Abbreviations: AF, atrial fibrillation; SII, systemic immune‐inflammation index; RFCA, Radiofrequency catheter ablation; BMI, body mass index; LAD, left anterior; LVEF, left ventricular ejection fraction; HbA1c, glycosylated hemoglobin; eGFR, estimated glomerular filtration rate; hs‐CRP, high sensitivity‐C reactive protein; OR, odds ratio; CI, confidence interval.Click here for additional data file.


The aim of this retrospective study was to analyze the effect and potential adverse effects of intravenous lipid emulsion (ILE) in poisoned dogs and cats over a 5 years period.Medical records of 313 dogs and 100 cats receiving ILE between 2016–2020 were analyzed for suspected toxicant, clinical signs, ILE dosages and frequency, the effect and adverse effects of ILE, and patient outcome.Dogs and cats were poisoned with mostly unidentified toxicants (48%), rodenticides (8%), recreational drugs and nuts (7% each) and other toxicants. Clinical signs included neurologic deficits (63%), cardiovascular signs (29%), thermoregulation (21%) or gastrointestinal abnormalities (17%). Treatment with ILE was initiated within a median of 6.0 h (1.0–91.0 h) after poisoning. Dogs and cats received a total amount of median 8.0 mL/kg (1.5–66.6 mL/kg) and 15.8 mL/kg (1.8–69.4 mL/kg) ILE, respectively. A positive effect was observed in 74% of the patients, whereas clinical signs worsened in 4% of the patients after ILE administration. No subjective effect was detected in 22% of the patients. Suspected or possible adverse effects of ILE occurred in 6% of the patients, including neurological signs (temporarily reduced consciousness and ataxia), bradycardia, hyperthermia, vomiting, diarrhea, respiratory distress, worsening of the general behavior, facial swelling, and thrombophlebitis. The overall survival rate was 96%. One dog who potentially experienced adverse events was euthanized.ILE treatment was successful in most patients but can be associated with adverse effects. Administration of ILE should be carefully selected on an individual basis after weighing the possible benefits against potential adverse effects. In addition to symptomatic treatment and decontamination attempts, intravenous lipid emulsions (ILE) are commonly administered to eliminate lipophilic toxicants from their sites of action. Several case reports have been published, but only one prospective clinical study  apart frThe first studies in the 1970s and 1980s described the effects of ILE on the pharmacokinetics of chlorpromazine  and ciclGiven the current knowledge gap on ILE therapy in VM, the objective of this retrospective study was to analyze the use of ILE in a large cohort of dogs and cats with confirmed or suspected poisonings, including the effects, adverse effects, applied dosages and outcomes of ILE. These cases were presented at the emergency service of a transregional veterinary referral center over a 5 years period. We hypothesized that the administration of ILE presents a safe treatment option and leads to a reduction in clinical signs of poisonings with lipophilic toxicants.2.Electronic medical records (MR) of the emergency service of the transregional veterinary referral center in Germany were searched for “poisoning”/“intoxication,” “intravenous lipid infusion,” or the brand of the available intravenous lipid infusion “Lipofundin” between 2016–2020.n = 405), transdermal (n = 6), or oral and transdermal (n = 2) poison exposure that received ILE  were included in the retrospective evaluation.Meeting these inclusion criteria, 416 patients were identified. Three patients with underlying primary neurologic diseases diagnosed in the course of the disease or with presumed poisoning but without evidence of poisoning based on the patient history or physical examination were excluded from the analysis. Thus, a total of 413 animals (313 dogs and 100 cats) with oral  demographic data; (2) clinical signs at admission and during hospitalization; (3) the toxicant ; (4) times from poisoning and presentation at the clinic to ILE application; (5) dosages of ILE bolus and continuous rate infusion (CRI); (6) duration of application, frequency, and the total amount of ILE administered to the patient ; (7) clinicopathologic findings, including hematology , biochemistry profile , and plasma ionized calcium concentration ; (8) the effect and adverse effects of ILE administration; (9) outcome, (10) disease course with any adverse effect, and (10) duration of hospitalization.p < 0.05.Patient data were recorded using a commercial software program  and were statistically analyzed . Continuous data were evaluated for normal distribution using the D’Agostino & Pearson normality test. Summary statistics were reported as medians and ranges (non-parametric data). The proportion of male and female animals as well as that of decontaminated and not decontaminated animals with or without adverse effects was analyzed using the Fisher’s exact test. ILE doses between dogs and cats with or without adverse effects and the times to improvement between dogs and cats were compared using the Mann–Whitney U test. The correlation between time to improvement and total ILE dose was analyzed using the Speaman correlation. Statistical significance was set at 3.3.1.Over the 5 years period, 313 dogs and 100 cats that presented with confirmed or suspected poisoning were treated with intravenous lipid emulsion (ILE). Predominant breeds in dogs were crossbreed  and Labrador Retriever , followed by Golden Retriever , Australian Shepherd , Jack Russell terrier , and Chihuahua . In cats, the Domestic Shorthair  was the predominant breed, followed by the British Shorthair  and crossbreed . Other breeds presenting with less than 3.0% of the species were not listed.p = 0.908). Sex was not documented in the electronic MR for one dog and one cat. The median age of all dogs included in the study was 3.6 years (0.2–14.1 years) and of all cats was 3.5 years (0.2–16.0 years). The median weight was 21.9 kg (1.3–64.0 kg) in dogs and 4.2 kg (0.8–8.5 kg) in cats.Male dogs  and cats  were represented with similar frequency as female dogs  and cats  and 2.0 h (0.1–72.0 h) in cats (n = 40) after suspected or witnessed toxicant ingestion or contact. Presentation at the emergence service of the clinic was within a median of 5.0 h (0.3–50.0 h) in dogs (n = 244) and 5 h (1.0–90.0 h) in cats (n = 43).Clinical signs became apparent within a median of 2.0 h (0.1–48.0 h) in dogs , cardiovascular signs or dehydration , altered thermoregulation (21.5%), and gastrointestinal signs . Respiratory signs , evidence of hemorrhage , and allergic reactions  were less common. In a few patients , no clinical signs were observed , followed by an altered general behavior . Poisonings with one or more than one toxicant were strongly suspected or observed with similar frequency . Poisonings with tremorgenic mycotoxins  and rodenticides  were more frequently reported than those with recreational drugs . These were followed in the frequency of occurrence by ingested toxic plants , food leftovers/kitchen waste , potentially tremorgenic mycotoxins , chemicals , antiparasitics , medication , feces- or manure-associated poisonings , nuts , and molluscicides . Some dogs  were poisoned with more than one toxicant in the same or even different toxicant group  was performed. However, the toxicant remained unidentified in almost half of the patients .Regardless of ILE therapy, most patients received supportive treatment, including intravenous fluids, analgesics, and antiemetics. Further decontamination and other elimination strategies  were also attempted in 244 patients prior to ILE administration  and 1.0 h (0.5–12.0 h) in cats (n = 97), respectively.Time from poisoning to first administration of ILE, reported in 243 dogs and 40 cats, was a median of 6.0 h (1.0–52.5 h) in dogs and 6.0 h (2.0–91.0 h) in cats. Time from presentation to first ILE administration was a median of 1.0 h (0.2–23.0 h) in dogs , and 26 animals had three ILE treatments . In a few patients, ILE was administered a fourth , fifth , or sixth time  or without an ILE bolus (n = 218) was given 307 times in a total of 240 dogs  and 106 times in 78 cats . The CRI was administrated at a median rate of 0.1 mL/kg/min (0.008–0.75 mL/kg/min) over a median of 45 min (15–240 min) in dogs and at 0.066 mL/kg/min (0.03–0.26 mL/kg/min) over 240 min (15–720 min) in cats.With up to six (dogs) or four (cats) administrations of ILE in an individual animal, a total of 199 boli of median 2.0 mL/kg (1.0–4.0 mL/kg) were given to 162 dogs  and a total of 49 2.0 mL/kg (1.5–2.8 mL/kg) boli to 42 cats . A continuous rate infusion (CRI) of ILE after the ILE bolus . In 76 dogs and 20 cats, additional ILE doses were administered. These patients received an additional two , three , four , five , or six  ILE boli or CRI. The second bolus (n = 32) or CRI (n = 52) was given at a median of 12 h (4–29 h) after the initial ILE administration, and additional ILE boli or CRIs were administered median every 12 h  in dogs and median every 12 h  in cats.About three-quarters of the patients received a single ILE dose  in dogs and 15.8 mL/kg (1.8–69.4 mL/kg) in cats, which was administered median 1 dose (1–6 doses) as boli and/or CRI in dogs and median 1 dose (1–4) doses as boli and/or CRI in cats .3.5.Laboratory findings (hematology and biochemistry profile) before and after ILE administration in dogs and cats are presented in 3.6.n = 234, 74.8%; cats: n = 54, 54.0%), was unchanged , or deteriorated  after ILE I. After ILE II, an improvement was observed in 64/76 dogs (84.2%) and 13/20 cats (65.0%), whereas 11 dogs (14.5%) and 7 cats (35.0%) were unchanged and 1 dog (1.3%) deteriorated. Overall, the administration of ILE was followed by a clinical improvement in 73.6% (n = 304) of the patients and a deterioration in 4.4% (n = 18). In 22% (n = 91) of the animals, ILE appeared to not have any substantial effect on the clinical signs  or reduced level of consciousness . Bradycardia , hyperthermia (dogs: n = 3), vomiting (dogs: n = 3), diarrhea (dogs: n = 3), respiratory distress , or deterioration of the general behavior (dogs: n = 3) were also recorded . Facial swelling as an allergic reaction (dogs: n = 1), ataxia (dogs: n = 1), and thrombophlebitis (cats: n = 1) were also observed .Adverse effects presumed to be associated with the administration of ILE occurred in 20/313 dogs (6.4%) and 4/100 cats (4.0%) in 24/413 patients (5.8%) after ILE administration . Such efp = 0.042). In cats, there was no significant difference between these two groups . One dog with suspected poisoning with an unknown toxicant experienced respiratory arrest and was euthanized. In all other patients, adverse effects of ILE therapy were transient, and these patients survived  without severe long-term consequences. Some patients were discharged despite still having mild signs of questionable adverse effects such as diarrhea (n = 2) after ingestion of diclofenac and THC or hyperthermia (n = 2) after ingestion of caffeine or nicotine.Adverse effects were observed after administration of a median of 11.9 mL ILE/kg (2.0–66.6 mL/kg), including ILE boli at a median of 1.5 mL/kg (2.1–2.0 mL/kg) in 12 dogs and a CRI at a median of 0.25 mL/kg/min (0.008–0.37 mL/kg/min) over median 60 min (20–720 min) in 17 dogs and 4 cats. A higher total amount of ILE was given to dogs that experienced adverse effects  than to dogs without adverse effects of ILE treatment .Few patients that experienced adverse events underwent decontamination efforts  before ILE administration. This included induced emesis (3/5), gastric lavage (1/5), and gastrointestinal lavage (1/5). Among those patients where decontamination was not previously attempted, some dogs had already vomited (4/19), and others received activated charcoal (7/19) or intestinal lavage (1/19). Omitting decontamination had a higher odds ratio (OR) for the development of adverse effects after ILE (n = 9), rodenticides, food leftovers/kitchen waste, or recreational drugs (n = 3 each), chemicals, toxic plants, medication, nuts, or antiparasitics  were hospitalized for a median of 1 day . Most  of those ILE-treated patients survived to discharge from the hospital . Thirteen patients (3.1%) were euthanized  and six patients (1.5%) died .Most patients . Dogs recovered faster  than cats . Time to improvement correlated mildly with ILE total dose in dogs , but not in cats (p = 0.925). Longer recovery times of 4–6 days occurred in dogs with walnut (n = 3), oleander, tetrahydrocannabinol (THC), permethrin, alpha-chloralose, or metronidazole poisoning, or exposure to an unidentified substance (each n = 1). During hospitalization, no clinical improvement could be documented in 18/413 patients , or a statement regarding the clinical improvement was not possible based on the patient medical records .Overall time to clinical improvement after presentation was a median 7.0 h  of the animals presented with poisonings during that time period (413/800), which is the largest series of dogs and cats with ILE treatment for poisonings compared to previous reports including less than 20 cats and 9 dogs, respectively .n = 24; cats: n = 30). Some of the affected animals presented in status epilepticus  or postictal state , while others were already sedated . These difficulties in controlling seizures, similar to paralysis and coma, can result in complications, such as aspiration pneumonia or renal injury carrying a high mortality rate. These cases are indications for the use of ILE if lipophilic toxicants are presumed or confirmed (p > 1.0 accumulate in lipophilic solvents as a result of the concentration gradient that is created between the lipophilic and hydrophilic phases (“lipid sink”), and can thus be removed from the tissue  by means of ILE therapy , THC (log p = 6.1), moxidectin (log p = 5.3), ivermectin (log p = 4.37), carprofen (log p = 4.09), ibuprofen (log p = 3.5), and lidocaine (log p = 1.81) are examples for substances that are suitable for removal via ILE therapyp = 0.12), ethylene glycol (log p = −1.36), or xylitol (log p = −2.56) poisoningp values such as ivermectin (p = 1.1) justifies the use of ILE for the treatment of metaldehyde poisoning. Assuming that metaldehyde is of rather low lipophilicity (with log p = 0.12), administration of ILE would presumably be ineffective with metaldehyde poisoning. Nevertheless, a beneficial effect has been reported in canine and feline metaldehyde poisoning for ILE combined with other supportive measures  in which post-treatment biochemical parameters were recorded allowing for a comparison of the values before and after treatment to evaluate the effect of ILE. Laboratory values important after ILE application, such as triglycerides or blood gas analysis, were not recorded. Thus, given the small number of patients, conclusions about an effect of ILE based on laboratory parameters is not possible.Early administration of ILE after toxicant ingestion is key for therapeutic success. In the present study, ILE treatment was initiated after a median of 6.0 h after ingestion of or contact with the toxicant, whereas the first ILE bolus or CRI was given at a median of 1.0 h after the presentation, showing that the delay was mostly due to the time from poisoning to presentation. Still, this approach allowed a fast recovery (within a few hours) in most cases, whereas longer recovery periods in dogs (up to 6 days) can be explained by neurotoxic effects and a postictal state .Generally, several different doses, dosing intervals, and durations between applications were used. ILE boli were mostly administered at 2.0 mL/kg in both dogs  and cats 20/49, 40.8%), whereas a dose of 1.5 mL/kg was administered in some dogs  as used or recommended by others , 27, 35., 40.8%, Most patients experienced treatment success as assessed based on the clinical status, after a single dose of ILE (76.8%), whereas clinical signs continued to improve after a second (17%), third (4.1%), or fourth to sixth ILE dose (2.2%) in some patients. Repeat dosing of ILE is not routinely performed, but appears safe (with ≤2 mL/kg per dose) and might offer additional benefit. However, our results support the recommendation to discontinue ILE treatment if an effect is not seen after 2–3 doses .50 in rats of 67 mL/kg  are reported in dogs and 15–18 mL/kg up to 31.5 mL/kg) in cats , 50, 51,.5 mL/kg 67 mL/kg . Howeverp = 3.79In this retrospective evaluation, ILE was given pre-emptively in some patients  which count towards the population of animals with an unchanged condition  after ILE administration. On the other hand, failure of ILE to have positive effects could also be explained by the lack of decontamination, representing another limitation of this retrospective evaluation. Additionally, in the absence of lipophilicity, toxicant elimination by ILE administration is not expected for toxicants such as α-chloralose, coumarin derivatives, metronidazole, and theobromine, and is questionable for macadamia nuts, walnuts, and metaldehyde. However, the decision to use ILE was at the discretion of the clinician and was often made based on the presence of severe neurological sings. Also, the contribution of simply time and physiological metabolism/excretion, unknown substances and doses exposed to in some cases, further supportive care, ILE administration, and/or in some cases a potential “lipid shuttle” created by the lipophilic drug propofol used for general anesthesia (log n = 20), or as “possible” for any effect that could have also been caused by the toxicant, but the patient did not show this clinical sign to the same extent prior to ILE therapy (n = 11). However, progressive clinical signs caused by the toxicant  cannot be reliably distinguished from the variance of clinical signs or possible sequelae  in all cases. Adverse effects of ILE considered as very likely occurred in a few cases (n = 12) of poisoning with an unidentified toxicant, rodenticides, food waste, chemicals, or plants. Progression of clinical signs of poisoning such as recumbency, comatose state, vomiting, dyspnea or apnea, somnolence, and bradycardia are also challenging to distinguish from signs of nonspecific worsening after ingestion of an unknown toxicant or without decontamination measures. For example, somnolence could have also been induced by recreational drugs  or hyperthermia by tremorgenic mycotoxins , recreational drug ingestion , or ingestion of food leftovers/kitchen waste containing caffeine (n = 1). Similarly, diarrhea could have been caused by ingestion of an NSAID  or recreational drug , sudden reduced consciousness and cardiovascular status may have been due to macadamia ingestion (n = 1), or progressive bradycardia resulting from permethrin ingestion (n = 1) rather than presenting adverse effects of ILE. Erring on the side of caution and minimizing the risk of side effect neglect, all clinical deteriorations that could have possibly been linked to ILE administration were assumed and included as such. Thus, while the study suggests ILE therapy to be successful in some cases, another important conclusion – given the presumably overestimated temporary side effects in about 6% of the patients – is that ILE appears safe in dogs and cats as adverse effects were not observed in >94% of ILE-treated dogs and cats. In line with this, standardized documentation of the clinical course is lacking due to the retrospective nature of this study, and medical record entries are more likely for adverse effects that occurred than steps of clinical improvement.Adverse effects of ILE were classified as “very probable” if occurring immediately after ILE administration (In veterinary medicine (VM), similar complications to human medicine (HM) are described for ILE treatment of poisoning and total parenteral nutrition (TPN). These include cardiovascular complications including asystole, cardiac arrest, pulmonary microembolism, and lower rates of returning spontaneous circulation after resuscitation . MaximuThe occurrence of lipid overload syndrome, however, not only depends on the infusion rate and total dose but is also affected by the composition of the lipid emulsion . Fat oven = 169) underscores the importance of these measures before initiation of ILE therapy (Finding that adverse effects of ILE occur predominantly (up to 79%) with the omission of prior decontamination  and clinical (electrocardiography (ECG) and blood pressure monitoring) as well as clinicopathologic variables  are now needed to establish evidence-based recommendations for ILE treatment and monitoring in dogs and cats. Some of these parameters were evaluated in this study, but conclusions are limited without controls. Useful information was provided for possible adverse effects of ILE. Given that many dogs and cats received ILE preemptively and standardized protocols for the treatment and monitoring of patients with presumed or confirmed exposure to hydrophobic poisoning agents were lacking, the treatment efficacy of ILE has to be carefully interpreted. Although the study suggests that ILE is safe to use in small animal patients, the possibility of adverse effects and limited evidence of clear benefits preclude the recommendation to use ILE routinely in the treatment of poisoning cases. Rather, its indication should be carefully considered on an individual basis.5.Reviewing 313 canine and 100 feline poisoning cases treated with ILE beneficial effects ascribed to the administration of ILE are presumed based on the clinical impression, but their quantification remains challenging. The clinical deterioration due to ILE adverse effects cannot be reliably differentiated from toxicant-associated complications, but the rate of adverse effects is suggested to be generally low (<6%). Lack of adverse effects in >94% of ILE-treated patients confirms a wide window of safety at the recommended doses (1.5 mL/kg bolus followed by 0.25 mL/kg/min over 30–60 min in dogs and cats or a CRI of 0.066 mL/kg/min over 240 min in cats). ILE-treated cases also had a high survival rate, but apnea requiring euthanasia in one case cannot be excluded as an adverse effect of ILE administration.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because Ethical review and approval was not required for the animal study because data were only retrospectively analyzed.CM: Conceptualization, Formal analysis, Data curation, Writing – original draft. RH: Formal analysis, Methodology, Validation, Writing – review & editing. DK: Data curation, formal analysis, review. RD: Formal analysis, Methodology, Validation, Writing – review & editing, Conceptualization, Funding acquisition, Project administration, Resources, Supervision.


Saroglitazar, a dual PPAR α/γ agonist, is useful in management of NAFLD and diabetic dyslipidemia. Here, we report the safety and efficacy of saroglitazar in NAFLD patients with or without diabetes including compensated cirrhosis.Patients, started on saroglitazar 4 mg were prospectively evaluated for 52 weeks in a tertiary care center in Eastern India. Effectiveness was measured in terms of anthropometric measurements, fasting blood glucose, LFT, lipid profile, HbA1c, and elastography parameters (LSM and CAP) measured at baseline, 24, and 52 weeks. Adverse drug reactions were monitored.P < 0.001). Significant reduction was also seen in CAP values, ALT, AST, HbA1c, LDL, total cholesterol, and triglyceride values. There was no significant weight change along the study interval. Pruritus occurred in one patient who required treatment discontinuation and another patient had mild symptomatic loose motion.A total of 112 patients were enrolled in the study, of whom 63 patients were taken up for per‐protocol analysis. Mean age was 49.11 ± 11.09 years and 46(73%) were male. Thirty‐four (54%) were nondiabetic. Eleven patients had compensated cirrhosis. There was significant improvement of LSM from baseline (11.03 ± 7.19 kPa) to 24‐week (9.29 ± 6.39 kPa) and 52‐week (8.59 ± 6.35 kPa) values respectively , were started on saroglitazar 4 mg. There was significant improvement of LSM from baseline (11.03 ± 7.19 kPa) to 24‐week (9.29 ± 6.39 kPa) and 52‐week (8.59 ± 6.35 kPa) values respectively. The favorable response was seen across all the fibrosis stages as categorized based on LSM values . Significant reduction was also seen in CAP, ALT, AST, HbA1c, LDL, total cholesterol, and triglyceride values. There was no significant weight change along the study interval. Pruritus occurred in one patient who required treatment discontinuation. Non‐alcoholic fatty liver disease (NAFLD) is a modern‐day epidemic. Its prevalence is almost 30% in Asian countries.Pathogenesis of NAFLD is multifactorial. NASH often develops in the context of excess adiposity and systemic insulin resistance.Lifestyle modification with exercise and achieving weight loss remains the cornerstone of therapy for NAFLD, but it is very difficult to sustain for a prolonged period. Despite better understanding of the pathogenesis and progression of NAFLD‐/NASH‐related liver disease, drug targeting key areas of metabolic pathways is lacking. In the last decade or so, multiple drugs have been tested for this condition, but till now a reliable medicine with a definite therapeutic end point is lacking. Also, in advanced stages of the disease, lifestyle modification alone is unlikely to be adequate management and may not be justifiable as monotherapy.Saroglitazar is a dual potent PPAR‐α/γ agonist. Synergistic effect of improved lipid oxidation and improved insulin resistance by PPAR‐α and PPAR‐γ, respectively, makes rational its use in NASH/NAFLD. Its efficacy in management of patients of NAFLD and diabetic dyslipidemia is being reported throughout the world.Diagnosis of baseline severity and assessment of therapeutic response of NAFLD have predominantly shifted to noninvasive methods. Liver biopsy, although considered gold standard, has its drawbacks, such as being invasive and less patient acceptance, particularly in repeated testing.We aimed to evaluate the real‐world safety and efficacy of saroglitazar 4 mg in patients with NAFLD primarily by seeing the improvement in liver steatosis and stiffness.This is an investigator‐initiated, single‐center, prospective, observational, open‐label, single‐arm study to evaluate the safety and efficacy of saroglitazar 4 mg in patients with NAFLD/NASH in real‐life setting, conducted in a tertiary care research institute in Kolkata.Ethical clearance was obtained from institutional ethical committee.The included patients were of age ≥18 years, and diagnosed for NAFLD fulfilling the AASLD guidelines,The patients with any evidence of alcoholic liver disease, significant alcohol use , concomitant use of any steatogenic drugs, and clinical or lab evidence of other liver disease have also been excluded from the study. Patients with decompensated cirrhosis have been excluded. Other liver illnesses like chronic hepatitis B or C infection, Wilson's disease, and drugs causing liver fibrosis like amiodarone, methotrexate, etc., have been excluded. Patients taking thiazolidine diones or saroglitazar in the last 6 months were also excluded.The patients found to have any confounding factors, which may overestimate the FibroScan values like liver congestion, ascites, liver inflammation due to any recent liver illness or drinking alcohol, benign or cancerous tumors in the liver, biliary obstructions, etc., were also excluded from the study.The person who has known allergy, sensitivity or intolerance to saroglitazar or formulation ingredients, women with pregnancy or lactation or of childbearing potential and not using appropriate contraceptive measures, has history or other evidence of severe illness or any other conditions that would make the patient, in the opinion of the investigator, unsuitable for the study and those who used vitamin E ≥ 800 IU/day or multivitamins containing vitamin E ≥ 800 IU/day in the 1 month preceding screening visit, were also excluded from the study.Saroglitazar 4 mg once daily was prescribed in routine clinical practice to eligible patients, who visited the outpatient department of AMRI Hospital, at Salt Lake, Kolkata in April 2019, and fulfilled the inclusion/exclusion criteria, along with routine care of diet and lifestyle modification. Informed consent was taken from every participant. The patients were followed up at an interval of 3 months and up to a total of 1‐year duration and the safety and efficacy data were collected in an excel sheet at every follow‐up visit. The data were collected by interviewing every participant using a set of questionnaires related to demographic features like age, gender, anthropometric measurement, family history for liver disease or other metabolic disorder, ongoing medications, detailed medical history, including personal history of alcohol intake, and any comorbidities like dyslipidemia, diabetes, and hypertension. A trained technical assistant did all the physical examination. Height was measured by a standard stadiometer, and weight was measured using a standard bathroom scale. Anthropometric measurements were cross‐checked to ensure inter‐observer reliability. Body mass index (BMI) was calculated using height in meter and weight in kg.Baseline patients demographics like age, gender, weight, height, waist circumference, co‐morbid conditions, and ongoing medications were recorded.Detailed medical history, including personal history of alcohol intake, concomitant medication, and presence of metabolic comorbidities like obesity, hypertension, diabetes mellitus, coronary artery, and cerebrovascular disease, has been recorded. Family history of any metabolic or liver‐related disorders has also been captured in the excel sheet at the baseline visit.2 and ≥ 30 kg/m2 respectively.The EchosensFibroScan® 530 Compact machine has been used to measure both fibrosis (liver stiffness) and steatosis (ultrasound attenuation rate/ CAP), together by an expert and Echosens certified technician. M and XL probes were used for subjects with BMI less than 30 kg/mAt baseline, patients were categorized into four categories based on liver stiffness (LSM) value: (i) F0–F1: LSM <7 kPa; (ii) F2: LSM 7–10 kPa; (iii) F3: LSM >10 to <14 KPa; and (iv) F4: LSM >14 kPa.Patients in the last group also underwent upper GI endoscopy for variceal screening.The patients on continued saroglitazar 4 mg once daily therapy have undergone follow‐up elastography at 24 and 52 weeks. Anthropometric measurements were taken at each clinical visit. Patients underwent testing for lipid profile, LFT, HbA1c, and CBC at baseline, 24 weeks, and 52 weeks. The data were analyzed using paired t‐test on SPSS Version 22 statistical package.At every follow‐up visit, safety parameters were assessed in the form of history regarding possible side effects as well as clinical examination.P values less than 0.05 were considered significant. All the statistical analysis was done on SPSS ver 22 statistical package.Continuous data with normal distribution were presented as mean and SD and without normal distribution as median and IQR. Normally distributed continuous data were analyzed using paired t‐test. Wilcoxon signed‐rank test was used for all skewed non‐normal data. All categorical data were presented as proportion and analyzed using the χ2 test. Between April and October 2019, 112 patients were screened for the study. Thirty‐six had one or more exclusion criteria, treatment had to be stopped in one due to allergic complication, 12 patients did not complete 12‐month therapy and were lost to follow‐up. Sixty‐three patients were taken up for per‐protocol analysis.Basic demographic profile of patients is shown in Table n = 55) and 81% (n = 52) respectively. Median LSM at baseline was 8.5 (4.2–35.6) kPa. Eleven patients had LSM >14 kPa, of them three were found to have esophageal varices. The median CAP value was 328 (233–400) dB/m.Baseline investigations are shown in Table Compared with baseline, after 24 and 52 weeks of saroglitazar therapy, there was reduction in ALT levels by 37% and 52% respectively, which was statistically significant. Similar trends were seen in AST also. There was statistically significant reduction in cholesterol and triglyceride levels at 52 by 24.1% and 40.6% (Table Eleven patients had LSM ≥14 kPa corresponding with F4 fibrosis.P < 0.001] and 52 weeks  as compared with the baseline [328 (46) dB/m] (Table There was a significant improvement in median CAP at 24 weeks  and at week 52  was also statistically significantly improved than the baseline median LSM of 8.5 (3.9) kPa (Table The median LSM at week 24 [6.5 (2.8) kPa, There was no significant weight reduction in the patient population, and BMI distribution was similar at baseline and 52 weeks (Table Saroglitazar 4 mg once daily was found to be safe and well tolerated, as there was no severe reported drug‐related major adverse event, which results in discontinuation of the therapy, except one case of minor itching, which subsided within 2 weeks of treatment discontinuation and one other case who has reported mild symptomatic loose motion.This study aimed to test the efficacy and tolerability of saroglitazar in an extended population including nondiabetics and ones having significant fibrosis on elastography. It is shown that saroglitazar shows significant reduction in LSM and CAP values as well as improvement in metabolic parameters. One novel finding is that saroglitazar is tolerable, safe, and efficacious in compensated F4 fibrosis also. There has been limited data regarding the efficacy of saroglitazar in the Indian population. Padole et al.This study adds to real‐world experience of saroglitazar in the NASH population. It is predominantly recommended for patients with diabetes, but Sarin et al. conducted a study including nondiabetics as well.As α effect of saroglitazar is more than γ agonism, lipid profile improvement is expected to be more than the improvement of glycemic status. It was corroborated in the result as well with patients having around 25% improvement in total cholesterol and LDL level and 40% reduction in triglyceride level. As expected from γ agonism, there was weight gain in the patient population despite improvement in LSM and CAP, which is shown in other studiesLiver enzymes have been used traditionally to assess the extent of liver injury. It has inherent limitations being nonspecific and not correlative with fibrosis. However, it is very useful in practical setting due to its availability and low cost. Study population showed a significant reduction of ALT and AST both at 24 and 52 weeks. It corroborates with recent real‐life dataPerhaps this study was the first of its kind to test the efficacy and safety of saroglitazar in compensated cirrhotics. Cirrhosis was diagnosed based on either LSM values or presence of varices on upper GI endoscopy with other causes being excluded. Saroglitazar was well tolerated with no new onset decompensation in this group as well as efficacious with approximately 23% reduction in LSM values at 52 weeks as compared with baseline. This is definitely a novel finding. Although only 11 patients were cirrhotics, further studies with larger patient population are warranted to possibly expand the use of this molecule.The first strength of the study is its patient population. Inclusion of nondiabetics and cirrhotics allowed to assess efficacy and safety of saroglitazar in this less studied population. The second strength is longer follow‐up duration of 52 weeks where most studies have shorter follow‐up data. The third strength is the use of noninvasive markers at frequent time interval that mimics real‐life experience, which most of the physicians follow.Limitation of this study remains that it cannot predict superiority over other molecules presently being researched for NASH. Absence of biopsy data would indicate that idea of baseline necro‐inflammatory activity was unknown. Third, follow‐up data were not there on whether discontinuation of drug would lead to reversal of biochemical and stiffness parameter improvement or persistence of benefit of drug. This very important question was not addressed in the study design. Fourth, cardiovascular outcome was not assessed. The cause of mortality and morbidity in NASH population is predominantly cardiovascular. So, whether improvement of metabolic parameter ultimately translates to improvement in cardiovascular outcome or not is a question that would require long‐term follow‐up, for which this study was not designed. Fifth, there was no placebo arm to compare the contribution of lifestyle modification alone to addition of saroglitazar.Saroglitazar is effective and safe in improving biochemical parameters and LSM and CAP values irrespective of weight reduction. It may be used in compensated cirrhotics with close monitoring for side effects.The study design was approved by institutional ethical committee of AMRI Hospitals, Salt Lake.Written consent was obtained from each participant after explaining in their own vernacular, consent forms preserved by the author.


Patients with higher AGEs accumulation presented decreased bone protein expression (sclerostin ; Dickkopf‐related protein 1 ; FGF‐23 ; and osteoprotegerin ), upregulation of the p53 gene, and downregulation of Dickkopf‐1 gene expression. Patients with high serum A1c levels presented greater cortical porosity and Mlt and reduced osteoblast surface/bone surface, eroded surface/bone surface, osteoclast surface/bone surface, mineral apposition rate, and adjusted area. Cortical thickness was negatively correlated with serum A1c  and pentosidine levels . AGEs accumulation in the bone of CKD patients was related to decreased bone protein expression, gene expression changes, and increased skeletal resistance to PTH; A1c and pentosidine levels were related to decreased cortical thickness; and A1c levels were related to increased cortical porosity and Mlt. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Advanced glycation end products (AGEs) accumulation may be involved in the progression of CKD‐bone disorders. We sought to determine the relationship between AGEs measured in the blood, skin, and bone with histomorphometry parameters, bone protein, gene expression, and serum biomarkers of bone metabolism in patients with CKD stages 3 to 5D patients. Serum levels of AGEs were estimated by pentosidine, glycated hemoglobin (A1c), and N‐carboxymethyl lysine (CML). The accumulation of AGEs in the skin was estimated from skin autofluorescence (SAF). Bone AGEs accumulation and multiligand receptor for AGEs (RAGEs) expression were evaluated by immunohistochemistry; bone samples were used to evaluate protein and gene expression and histomorphometric analysis. Data are from 86 patients . Median serum levels of pentosidine, CML, A1c, and SAF were 71.6 pmol/mL, 15.2 ng/mL, 5.4%, and 3.05 arbitrary units, respectively. AGEs covered 3.92% of trabecular bone and 5.42% of the cortical bone surface, whereas RAGEs were expressed in 0.7% and 0.83% of trabecular and cortical bone surfaces, respectively. AGEs accumulation in bone was inversely related to serum receptor activator of NF‐κB ligand/parathyroid hormone (PTH) ratio (R = −0.25;  Mineral and bone disorder (MBD) is a major complication of chronic kidney disease (CKD) and causes systemic effects, resulting in cardiovascular disease, bone fractures, and increased mortality.4, 5 Advanced glycation end products (AGEs) constitute one group of uremic toxins, whose effect on bone metabolism in CKD patients is poorly understood.MBD pathophysiology is related to the accumulation of many uremic toxins, such as phosphate and parathormone.6, 7AGEs represent a heterogeneous group of molecules, constituted by nonenzymatic glycation reactions reducing sugars, amino acids, lipids, or DNA. These AGEs molecules accumulate with the CKD progression and activate intracellular signals through nonspecific, specific receptors (RAGEs) and non‐receptor‐mediated mechanisms, leading to increased production of reactive oxygen species and inflammatory cytokines.11 Some studies have identified a link between AGEs, osteoporosis, and bone fractures in clinical settings.At the cellular level, AGEs are related to the decreased differentiation and proliferation of osteoblasts, osteoclasts, and mesenchymal stem cell apoptosis. AGEs also affect matrix protein production and lead to collagen cross‐linking activity alterations.14 Som Chen et al. tested the effects of AGEs lowering drug ALT‐711 in the aorta and bone. They observed bone AGEs content reduction without any improvement in bone mechanics. In humans, Mitome et al. observed a significant presence of pentosidine in bone from patients on dialysis. The pentosidine concentration was inversely related to the bone formation rate and volume.However, evidence of the effects in CKD is scarce, and the underlying mechanisms are not fully understood. In a rat model of renal osteodystrophy induced by adenine, Aoki et al. observed a greater accumulation of AGEs in peritrabecular osteoblasts and suppressed expression of runt‐related transcription factor 2 (RUNX2), alkaline phosphatase, secreted phosphoprotein‐1, and lysyl oxidase mRNA levels than in normal animals. The authors suggest that these findings represent suppression of osteoblast differentiation and function.9 ChenTogether, these studies reveal the need to deepen knowledge of the effects of AGEs on bone metabolism to generate new hypotheses to better understand their contribution to the pathophysiology of CKD‐MBD. For this reason, we performed an extensive examination of the impact of AGEs in bone from patients with CKD at different stages and treatments. Our working hypothesis is that CKD is associated with the accumulation of AGEs, which in turn results in the dysfunction of bone metabolism, expressed by bone morphological alterations, reduced protein synthesis, and changes in gene expression. Our primary aim was to identify and quantify the accumulation of AGEs and RAGEs in bone and AGEs in the blood  and skin and then study the relations between AGEs accumulation, bone histology, protein and gene expression, and serum markers of bone metabolism.n = 26), hemodialysis , and peritoneal dialysis . Patients were selected for convenience, sequentially, and according to the established inclusion and exclusion criteria. They were not part of another study, and all tests performed were part of the research protocol provided for this study.Eighty‐six patients at different CKD stages were enrolled in this observational and double‐center study from February 2016 to November 2017. Patients were recruited from the Nephrology Department's outpatient clinics at the Hospital de Clínicas of the State University of Campinas (UNICAMP) and São Paulo State University (UNESP) and divided into the following CKD subgroups: CKD stages 3–5 nondialysis on conservative management but still without a clinical indication for dialysis , and, specifically for patients under HD/PD, to be under these treatments for at least 3 months. The CKD‐EPI equation estimated the glomerular filtration rate. Patients in the HD subgroup were on chronic HD treatment three times weekly, 4 hours/session, using high‐flux and high‐efficiency polysulfone dialyzers. Patients in the PD subgroup were on automated PD (n = 18) or continuous ambulatory PD (n = 10).The inclusion criteria were age over 18 years, in CKD stages 3 to 5D according to  (KDIGO),19 andExclusion criteria were the presence of chronic inflammatory disease, primary hyperparathyroidism, kidney transplantation, acute cardiovascular event in the 3 months before screening for inclusion, cognitive impairment, cancer, HIV, and clinical instability; 12 (46%) patients in the CKD 3–5 nondialysis subgroup were classified as having CKD stage 3, 11 (42%) stage 4, and three (12%) stage 5 nondialysis. Written informed consent was obtained from all patients; the local ethics committee approved the study protocol under numbers CAAE 38108314.6.0000.5404‐45943115.9.0000.5404‐45777015.5.0000.5404, and the clinical and research activities being reported are consistent with the Declaration of Helsinki.AGE skin deposition was evaluated by SAF using the AGE‐Reader™ . This device measures fluorescence emitted from the skin influenced by the deposition of AGEs, and it calculates the ratio between emitted and reflected excitation light. The measurements were in triplicate on the ventral side of the forearm. Areas with arterial–venous fistulas, scars, and tattoos were avoided. The mean values were used for all statistical analyses and AGEs levels in skin expressed as arbitrary units (AU).In accordance with the manufacturer's instructions, serum pentosidine and N‐carboxymethyl lysine levels were determined by enzyme‐linked immunosorbent assay (ELISA) . The detection ranges of the pentosidine and N‐carboxymethyl lysine kits were 25–2000 pmol/mL and 5–100 ng/mL, respectively.Serum intact parathyroid hormone (PTH) levels (reference range: 15–65 pg/mL) were measured using a chemiluminescence assay . Serum 25‐hydroxyvitamin D levels (reference range: 30–100 ng/dL) were measured using a chemiluminescence method. Alkaline phosphatase (reference range: 30–120 IU/L) was measured using a kinetic colorimetric test . Serum calcium and phosphate levels and a general serum biochemistry profile were assayed by standard autoanalyzer techniques in an on‐site biochemistry laboratory . Serum tartrate‐resistant acid phosphatase 5b (TRACP‐5b) levels were determined using an ELISA kit , normal range: 1.2–6.7 U/L; sclerostin was assessed by Teco Sclerostin EIA Kit . Intact fibroblast growth factor 23 (FGF‐23), Dickkopf‐related protein 1 (DKK1), and receptor activator of nuclear factor kappa‐B ligand (RANKL) were measured using a multiplex assay kit . Plasma specimens were prepared for analysis utilizing a multiplex assay kit  according to specific protocols provided by the company. Blood samples were collected on a previously scheduled date for patients in the CKD 3–5 nondialysis and PD groups. Blood samples of hemodialysis patients were collected immediately before the week's second session.Bone biopsy samples were obtained from the right or left iliac crests through a trephine for bone biopsy (diameter = 7 mm), adapted to an electrical drill . A double labeling tetracycline course for 3 days (20 mg/kg/d), with a 10‐day interval, was used. The biopsy was performed 3–5 days after the last tetracycline dose. A bone fragment was divided into three parts for histomorphometry, immunohistochemistry, and molecular biology studies. The undecalcified bone fragments were submitted to histological processing and analysis. Bone histomorphometry was performed through a semiautomatic method . The static, dynamic, and structural histomorphometric indices were reported using international nomenclature.21 In brief, two adjacent 5‐μm sections of bone tissue were placed side by side on each slide. Bone sections were deacrylated in a 1:1 mixture of xylene and chloroform for 30 minutes, rehydrated in graded alcohol solutions, submitted to a quick semidecalcification with 1% acetic acid for 10 minutes, and rinsed twice with distilled water. Endogenous peroxidase activity was inhibited by a mixture of 3% hydrogen peroxide in methanol for 30 minutes, followed by two water washes. The samples were incubated with protein block  to block nonspecific binding. Sections were incubated overnight at 4°C in a humidified chamber using the primary rabbit polyclonal antibodies anti‐AGE  (dilution 1:5000) and anti‐RAGE  (dilution 1:200). After incubation with the secondary antibody, the slides were incubated with the avidin/biotin HRP complex. The revelation was developed with a Vector kit  (1 drop DAB +1 mL of the substrate). The sections were rinsed in distilled water and counterstained with Mayer's hemalum solution . Negative controls were performed by omitting the primary antibody. For analysis, the images were captured using an Olympus BX53 photomicroscope  integrated into a computer and analyzed using Image‐Pro Premier® software . The entire extent of the trabecular and cortical bone tissue regions were photographed with final magnifications of ×100 and ×400, and through the software, the areas of interest imunostained for anti‐AGEs and anti‐RAGEs were automatically quantified through the creation of a macro capable of identifying in each pixel of the image the shade of the brown color defined as being representative of positive immunostaining, in relation to the negative control. The immunopositivity was expressed as a percentage of the total software‐classified areas.Immunohistochemical quantification of the accumulation of AGEs and RAGEs expression in bone was performed by adapting a method previously reported by Gomes.22 In Protein lysates were extracted from bone samples. The bone contents of sclerostin, osteocalcin, DKK1, and FGF‐23 were measured with a multiplex assay kit  based on Luminex™ xMAP technology according to the manufacturer's instructions.After total RNA extraction from bone sample using trizol, a NanoDrop 1000 spectrophotometer (Thermo Scientific) was used to determine the total RNA amount. cDNA was synthesized from total RNA by reverse transcriptase  using a thermocycler .Gene expression was determined from the cDNA through quantitative PCR using SYBR Green  and a Rotor‐Gene Q thermocycler . The genes analyzed were SOST (AF_331844.1), RANKL (NM_003701.3), OPG (U94332), β‐catenin (X_87838.1), FGF‐23 (NM_020638.2), p53 (NM_001276760), DKK (NM_012242.4), Osterix (AF477981), ALP‐1 (J04948.1), collagen 1 (D21337.1), BGLAP (NM_199173), and the reference gene GAPDH  with their respective primers designed from IDT . Gene expression was calculated by the ΔΔCt method of relative quantification. Values are expressed as a multiple (fold) of the expression compared to the value of the calibrator. The statistical analysis between groups was performed using REST™ software .t test, the Mann–Whitney test, and the chi‐square test. To detect associations between AGEs accumulation and changes in bone metabolism, the median value of AGEs accumulation parameters  was used for the purpose of comparison. Spearman's coefficient test provided the significance of the correlation between noncontinuous variables. Statistical analyses were performed using SPSS 22.0 . A two‐sided p‐value <0.05 was considered statistically significant.The continuous variables are reported as the mean ± SD or medians and interquartile intervals. Categorical data are reported as frequencies and percentages. Comparisons between the continuous variables, skewed data, and categorical variables were performed using the Student's 2. The baseline characteristics of the study population are summarized in Table We considered data from 86 individuals for analysis; this was a population with a mean age of 51 ± 13 years; 48 (56%) were male, 41 (48%) were Caucasian, and 16 (19%) had type 2 diabetes. All participants had CKD; 32 (37%) were on HD, 28 (33%) were on PD, and 26 (30%) were on conservative management, displaying an estimated glomerular filtration rate of 26.9 (17.2–34.5) mL/min/1.73 mAGEs were detected in blood, skin, and bone in all patients. No correlation was found between measurements of AGEs in blood, skin, and bone.p = 0.0001) and dialysis vintage .In serum, the median levels of pentosidine, N‐carboxymethyl lysine, and glycated hemoglobin were 71.6 (44.2–121.2) pmol/mL, 15.2 (9.7–32.4) ng/mL, and 5.4% (5–6.1%), respectively. In skin, the median value of SAF was 3.05 (2.5–3.4) AU and was positively correlated with age .In bone, accumulation of AGEs and expression of RAGEs were detected in both trabecular and cortical surfaces in all patients. AGEs in trabecular and cortical bone covered 3.92% 1.6–15.3%) and 5.42% (3–12.1%) of its surface, respectively. RAGE expression in trabecular and cortical bone covered 0.7% (0.13%–2.88%) and 0.83% (0.2–2.3%) of its surface, respectively  and dialysis vintage  and negatively correlated with the serum RANKL/PTH ratio .AGEs in trabecular bone were positively correlated with AGEs in cortical bone , dialysis vintage , phosphate , and parathormone  and negatively correlated with serum glycated hemoglobin levels  and the TRACP‐5b/PTH ratio .RAGEs expression in trabecular bone was positively correlated with RAGEs expression in cortical bone  vs. 19.1 (9.8–34.7); p = 0.03], reduced osteoblast surface/bone surface , eroded surface/bone surface , osteoclast surface/bone surface , mineral apposition rate  and adjusted apposition rate/bone area . Cortical thickness was negatively correlated with serum glycated hemoglobin  and with pentosidine levels . No differences were observed in bone parameters according to median levels of N‐carboxymethyl lysine.Patients presenting high serum glycated hemoglobin levels displayed greater cortical porosity , DKK1 , FGF‐23  and osteoprotegerin  compared with patients presenting low trabecular bone AGEs levels  vs. 118 (107–136) μm; p = 0.003], trabecular number , and fibrosis volume/bone volume , and decreased trabecular separation  (Table Patients above the median of trabecular RAGEs expression had increased bone volume/tissue volume [26.2% (19.2–30.7%) vs. 18.9% (15.4–24.4%); p = 0.01) and FGF‐23 .Bone proteins and gene expression did not reveal differences according to the median trabecular bone RAGEs expression Table . PatientTo evaluate differences across treatment regimens regarding AGEs levels, we compared individuals on HD, PD, and conservative treatment. Only a few differences were noted between the treatment regimens.p = 0.009). Serum glycated hemoglobin levels were higher in HD than in PD and conservative treatment . RAGEs expression in trabecular bone was higher in HD than in conservative treatment , whereas no differences across groups were reported for cortical bone. No difference was found among groups regarding serum pentosidine levels, AGEs in skin, AGEs in bone surface, and RAGEs expression in cortical bone.N‐Carboxymethyl lysine levels were higher in PD patients than HD and conservative patients /bone volume (BV) and MAR, but the number of subjects in this analysis was limited to 10.As far as we know, this is the first study to reveal bone AGEs accumulation along CKD stages and treatments and its impact on histology, bone protein, and gene expression. In animals with CKD, Aoki et al. detected AGEs in peritrabecular osteoblasts by immunohistochemistry and western blot techniques.9 In h Sclerostin and DKK1 are Wnt/β‐catenin pathway inhibitors and are considered negative regulators of bone mass. Sclerostin is produced mainly by osteocytes, while DKK1 is mainly produced by osteoblasts. It is worth highlighting that bone AGEs accumulation was detected mainly around osteocytes and osteoblasts  ratio as an index that reflects bone responsiveness to PTH.35 In Our study found no correlation between serum, skin, and bone levels of AGEs. This finding agrees with previous observations; in human body tissues, organs and structures seem to present different affinities for AGEs accumulation, either by constitution itself as due to metabolism particularities. For example, the proteins in the human eye are highly susceptible to the formation of AGEs, which accumulate at a higher rate in diseases such as cataracts. As bone turnover is lower than other tissues, some authors hypothesized that bone was potentially more susceptible to AGEs accumulation and effects. AGEs measurement in serum samples remains a challenge because of the lack of standardized methods and because circulating AGEs may not accurately reflect their accumulation in body tissues, which results from long‐term exposure. Skin measurement using SAF attenuates this effect, since AGEs accumulation in the skin may be more closely related to AGEs deposition in the bone, yet the differences affect the correlation between these events in the intracellular synthesis of AGEs that vary across tissues.39 However, Sroga et al. observed differences in the progression of bone pathologies related to protein glycation by different sugars: in vitro glycation of bone using glucose leads to the formation of lower levels of AGEs, whereas ribosylation appears to support a pathway toward pentosidine formation. This observation suggests differential actions of different AGEs in cortical bone.Our results showed that serum pentosidine and glycated hemoglobin levels were related to decreased cortical thickness, increased cortical porosity, and mineralization lag time. We observed that glycated hemoglobin affected cortical bone differently than pentosidine. Previous studies in animals and humans showed direct relationships between glycated hemoglobin and cortical microarchitecture alteration.41 HowThis study had limitations. It was an observational and, essentially, descriptive study able to generate new hypotheses. The conclusions suggest relationships between bone AGEs accumulation and a decrease in essential bone proteins, changes in bone gene expression, potential increased skeletal resistance to PTH, and negative effects of serum AGEs on cortical bone. To overcome the lack of a control group, we attempted to perform statistical analyses through intragroup comparisons of AGEs accumulation parameters according to their respective medians; since there is no standardization of serum pentosidine and CML levels measurements at CKD setting, as well comparisons with more accurate methods such as HPLC, the results on these parameters must be interpreted with caution; we cannot exclude the possibility that the lack of correlation between AGEs accumulation in trabecular bone and BFR/bone surface (BS) was modified by serum PTH levels due to potential sample selection bias. Further interventional studies are required to confirm the mechanisms involved in these associations.Our study also had some strengths. Most importantly, (i) it demonstrated, using bone biopsy, that AGEs and RAGEs accumulate in the bone of CKD subjects; (ii) a myriad of morphofunctional and genetic parameters was analyzed, providing insightful data on the mechanisms involved in renal osteodystrophy; and (iii) skin, serum, and bone AGEs levels were evaluated, and their associations with bone metabolism impairment were explored. Pharmacological studies or dietary approaches should be proposed to test the effects of lowering AGEs on outcomes involving skeletal disease caused by CKD.We demonstrated that bone accumulation of AGEs occurs in patients with CKD and might affect the metabolism of this tissue. A possible mechanism would be a reduction in the synthesis of essential bone proteins and changes in gene expression. Cortical bone seems to be affected by different serum AGEs. The mechanisms behind these interactions, including differential effects according to AGEs types, should be explored in specific studies. Reducing AGEs accumulation may be a therapeutic target that would modify the progression of skeletal disorders in patients with CKD.10 PhaKélcia R. S. Quadros: Conceptualization; data curation; formal analysis; investigation; writing – original draft; writing – review and editing. Noemí A. V. Roza: Data curation; formal analysis; methodology; writing – review and editing. Renata A. França: Investigation; writing – review and editing. André B. A. Esteves: Investigation; writing – review and editing. Joaquim Barreto: Formal analysis; methodology; writing – original draft; writing – review and editing. Wagner V. Domingues: Formal analysis; methodology; writing – review and editing. Luzia N. S. Furukawa: Formal analysis; methodology; writing – review and editing. Jacqueline Teixeira Caramori: Investigation; writing – review and editing. Andrei C. Sposito: Conceptualization; writing – review and editing. Rodrigo Bueno de Oliveira: Conceptualization; data curation; formal analysis; funding acquisition; project administration; resources; supervision; writing – original draft; writing – review and editing.The authors declare there are no competing financial interests.https://publons.com/publon/10.1002/jbm4.10727.The peer review history for this article is available at Table S1. Clinical, demographic, and biochemistry findings of all CKD populations and subgroups.Table S2. Comparisons of bone gene expression according to median of accumulation of AGEs in skin, trabecular bone, and expression of RAGEs in trabecular bone.Click here for additional data file.


The aim of the study was to identify predictors for 3‐month poor functional outcome or death after aSAH and develop precise and easy‐to‐use nomogram models.The study was performed at the department of neurology emergency in Beijing Tiantan Hospital. A total of 310 aSAH patients were enrolled between October 2020 and September 2021 as a derivation cohort, while a total of 208 patients were admitted from October 2021 to March 2022 as an external validation cohort. Clinical outcomes included poor functional outcome defined as modified Rankin Scale score (mRS) of 4–6 or all‐cause death at 3 months. Least absolute shrinkage and selection operator (LASSO) analysis, as well as multivariable regression analysis, were applied to select independent variables associated with poor functional outcome or death and then to construct two nomogram models. Model performance were evaluated through discrimination, calibration, and clinical usefulness in both derivation cohort and external validation cohort.The nomogram model to predict poor functional outcome included seven predictors: age, heart rate, Hunt‐Hess grade on admission, lymphocyte, C‐reactive protein (CRP), platelet, and direct bilirubin levels. It demonstrated high discrimination ability , satisfactory calibration curve, and good clinical usefulness. Similarly, the nomogram model combining age, neutrophil, lymphocyte, CRP, aspartate aminotransferase (AST) levels, and treatment methods to predict all‐cause death also revealed excellent discrimination ability , satisfactory calibration curve, and clinical effectiveness. Internal validation showed the bias‐corrected C‐index for poor functional outcome and death was 0.827 and 0.927, respectively. When applied to the external validation dataset, both two nomogram models exhibited high discrimination capacity , good calibration ability, and clinical usefulness.Nomogram models constructed for predicting 3‐month poor functional outcome or death after aSAH are precise and easily applicable, which can help physicians to identify patients at risk, guide decision‐making, and provide new directions for future studies to explore the novel treatment targets. We constructed precise and easily applicable nomogram models to predict 3‐month poor functional outcome or death after aSAH. These prediction models can help physicians to identify patients at risk, guide decision‐making, and provide new directions for future studies to explore the novel treatment targets. The Hunt‐Hess grade, WFNS grade, and modified Fisher grade have been widely used to predict outcomes and help make clinical decisions.Therefore, based on this modern‐day, observational cohort, we aimed to identify new predictors for 3‐month poor functional outcome or all‐cause death and develop two novel nomogram models.22.1The study was a prospective, observational cohort study of aSAH patients admitted to the department of neurology emergency in Beijing Tiantan Hospital. The derivation cohort comprised eligible aSAH patients admitted between October 2020 and September 2021, while the external validation cohort was composed of aSAH patients enrolled from October 2021 to March 2022. The study was performed in compliance with the ethical guidelines from the Helsinki Declaration and was approved by the Institutional Review Board of Beijing Tiantan Hospital. Written informed consent was obtained from all the participants or their legal proxies. The inclusion criteria were (1) age of 18 years or older, (2) spontaneous SAH diagnosed via head CT scan, (3) presence of intracranial aneurysm confirmed by CT angiopathy (CTA), or digital subtraction angiopathy (DSA). The exclusion criteria encompassed: (1) SAH caused by other reasons, such as trauma, cerebral arteriovenous malformations, moyamoya disease, and intracranial tumors, (2) previous history of cerebral infarction, intracranial hemorrhage, vascular anomalies, and malformations, (3) acute kidney injury or chronic kidney disease, (4) concurrent systemic comorbidities including malignancy and liver cirrhosis, (5) premorbid modified Rankin Scale (mRS) >1.2.2Clinical information including demographic data (sex and age), previous history , neurological status, radiological characteristics, and laboratory tests on admission were all collected. Neurological status on admission were assessed by Hunt‐Hess grade and World Federation of Neurosurgical Societies (WFNS) grade. Radiological characteristics included modified Fisher grade, the number (“single” or “multiple”) of aneurysms, the location  of the aneurysms,Blood samples were drawn from an antecubital vein within 30 min upon arrival and prior to any treatment performed. Laboratory tests, including white blood cell (WBC), lymphocyte, monocyte, neutrophil, red blood cell (RBC), hemoglobin (Hb), platelet (PLT), C‐reaction protein (CRP), fibrin degeneration products (FDP), D‐dimer, fibrinogen (Fbg), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), potassium, sodium, chlorine, glucose, blood urea nitrogen (BUN), creatinine (Cr), estimated glomerular filtration rate (eGFR), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIl), creatine kinase isoenzyme MB (CK‐MB), and cardiac troponin I (cTNI), were collected.Treatment modalities, including surgical clipping, endovascular coiling, or conservative treatment, were determined by the professional and experienced endovascular specialists and neurosurgeons according to the condition of each patient under the guidance of the current guidelines.All treatment methods were not at the discretion of our researchers, and patients in our study were not predefined to a certain group of an intervention or exposure. Also, there was no experimental therapy performed on enrolled patients and no biospecimens were additionally extracted or retained for further investigation. Therefore, our study was a purely observational study with no‐trial designs. According to the clinical registration requirements recommended by the International Committee Medical Journal Editors (ICMJE), a purely observational study does not require to register.2.3All the patients were followed up through telephone interviews at 3 months after symptom onset by trained interviewers who were blinded to baseline characteristics and prognostic factors. Clinical outcomes included unfavorable functional outcome defined as mRS score of 4–6 or all‐cause death at 3 months.2.4Currently, the events per variable (EPV) criterion, especially an EPV of 10, is widely used as the lowest limit for logistics regression models to predict a binary outcome.2.5https://www.r‐project.org/, version 4.1.2). Shapiro–Wilk test was conducted to check whether the included variables were normally distributed. Continuous variables are expressed as means ± SD or medians with interquartile range according to the distribution of the data. Categorical variables are described as numbers (percentages).Statistics analysis was performed using IBM SPSS Statistics for Windows, version 22.0  and R software  curve (AUC) was applied to evaluate the discrimination capacity of the nomogram models. DeLong's test was performed to compare the discriminative capacity between the nomogram models and the core SAHIT model. Calibration curves were plotted to show the agreement between the nomogram models and the expected observation. A plot near the 45‐degree line would implicate the perfect calibration ability of the model. Furthermore, decision curve analysis was performed to assess the clinical effectiveness of the models by calculating the net benefits at different threshold possibilities.p‐values <0.05 were considered statistically significant.We used the “Matrix” and “glmnet” package to generate the LASSO regression results. The “SparseM,” “ggplot2,” “Formula,” “survival,” “lattice,” “Hmisc,” and “rms” package were applied to build nomogram models, generate the C‐index and calibration curves. In addition, the “pROC” and “rmda” package were performed to plot ROC curves and decision curves, respectively. All tests of significance were two‐tailed, and 33.1A total of 310 patients diagnosed with aSAH were included in derivation cohort. Among them, 64.5% (200) were females and the mean age was 56.1 ± 12.4 years old. Unfavorable functional outcome after 3 months was observed in 17.1% of the patients, while 3‐month all‐cause death was found in 9.0% of the patients. The baseline characteristics of patients in derivation cohort grouped by 3‐month poor functional outcome or all‐cause death are shown in Table 3.2Least absolute shrinkage and selection operator regression analysis was applied to select the potential predictors. In terms of 3‐month poor functional outcome, an optimal λ of 0.026 was adopted based on the minimum error criterion Figure . Therefop < 0.001], heart rate , Hunt‐Hess grade , lymphocyte , platelet , CRP , and DBIL  were all significantly associated with 3‐month poor functional outcome  = 0.093 (0.018–0.497), p = 0.005], neutrophil , CRP , AST , and endovascular coiling   = 1.070 1.035–1.105), 5–1.105, Based on the result of multivariable logistics regression analysis, two nomogram models were constructed to better visualize the estimation of the individual risk for poor functional outcome or all‐cause death Figure . Each pr3.3p = 0.002)   than that of the core SAHIT model  Figure . In term) Figure . Further) Figure .The two nomogram models were internally validated by 1000 bootstraps validation method. The bias‐corrected C index for predicting 3‐month poor functional outcome and all‐cause death was 0.827 and 0.927, respectively, which all exhibited excellent discrimination capacities.External validation of two nomogram models was also conducted. A total of 208 eligible aSAH patients were included in the external validation cohort. The majority of the patients were female (63%) with a mean age of 56.8 years old. Poor functional outcome after 3 months was observed in 19.7% of the patients, while the mortality rate was 10.6%. No significant difference was identified between the derivation cohort and external validation cohort Table . The res4In the present study, we developed and validated two novel nomogram models for predicting poor functional outcome or all‐cause death at 3 months in patients with aSAH. Both two models demonstrated favorable performance and predictive accuracy in derivation and externally validation cohorts.4.1A total of seven pronounced risk factors, including age, Hunt‐Hess grade, heart rate at presentation, lymphocyte, CRP, platelet, and DBIL levels, were finally incorporated into the nomogram model to predict 3‐month poor functional outcome. Age was reported to be a major significant predictor for poor outcome in many literatures.4.2Variables associated with all‐cause death at 3 months after aSAH onset are partially overlapped with those associated with disability, such as lymphocyte and CRP, which explains the shared pathogenesis of death and disability after aSAH. Additional parameters important for death prediction include neutrophil, AST levels and treatment methods. Aneurysmal bleeding promotes neuroinflammatory responses, which is characterized by elevated neutrophil counts.When it comes to treatment methods, our study has demonstrated that endovascular coiling was associated with patient survival. As it known to all, surgical treatment can significantly improve the prognosis of patients with aSAH compared with conservative treatment. However, how to choose between endovascular coiling and surgical clipping remains controversial. The International Subarachnoid Aneurysm Trial (ISAT) compared the safety and effectiveness of endovascular coiling and surgical clipping in 2143 patients with ruptured aneurysms.4.3Recently, Jaja et al. developed the SAHIT prediction models for poor functional outcome or death after 3 months in patients with aSAH.4.4There are several strengths in the current study. First, the identified parameters not only can be readily measured and obtained from routine examinations but also convenient to use in acute aSAH phase without the need for much experience to evaluate imaging scales and consider the subsequent complications during treatment course. Second, the LASSO regression applied in our study has advantages to lower data dimensionality and reduce multicollinearity between variables, thus, it is performed well in selecting the potential significant parameters. Moreover, nomogram excels in simplicity over traditional analysis utilizing odd ratios and can help to better visualize the model to generate an individual risk of poor outcomes, making it beneficial for physicians to apply in clinical practice.However, there are also some limitations in our study to be recognized. First, our study was conducted in a single tertiary center with a relatively small sample size, which might introduce selection bias. A multicenter and large study is required to validate our findings in the future. Second, we only evaluated outcomes at 3 months after symptom onset in patients with aSAH, future studies should focus on the long‐term prognosis of aSAH patients. Moreover, external validation from a different cohort is still needed to further verify the generalizability of our models.4.5The nomogram prediction models in the present study could assist in providing patient‐centered information of poor prognosis and help clinicians to communicate effectively with patients and their relatives. More importantly, using the readily obtained variables on admission, our prediction models help meet the demand for identifying and screening patients at high risk of poor outcomes quickly at an early stage of aSAH. Therefore, clinicians can pay more attention to these high‐risk patients and promptly conduct personalized therapy to prevent further deterioration and significantly improve prognosis of these patients. Furthermore, another potential clinical significance of our study is that the nomogram models contain predictors that can reflect the process of early brain injury. Hence, it provides meaningful insight into future studies to explore treatments that target neuroinflammatory response, microthrombosis formation, toxic effects of degradation products and so on.5In the present study, nomogram models constructed for predicting 3‐month poor functional outcome or death after aSAH demonstrated good discrimination, accuracy, and clinical usefulness. The predictor items in these two nomogram models are not only easy to obtain upon admission but also can reflect the pathogenesis of aSAH to a certain extent. Therefore, these two models can help physicians to identify patients at risk, guide decision‐making at an early stage, and provide new directions for future studies to explore the novel treatment targets.SL collected the original data, performed the statistics analysis, interpreted the data, and drafted the original manuscript. JZ, NL, and DW contributed to data interpretation. XZ designed the research and handled funding and supervision. All authors read and approved the final manuscript.This study was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019‐I2M‐5‐029), Beijing Municipal Committee of Science and Technology (Z201100005620010), Beijing Hospitals Authority Innovation Studio of Young Staff Funding Support (code: 202112), and the Ministry of Finance of the People's Republic of China .None.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.


Coronary angina due to low estrogen is relatively common around menopause, with almost no reports associated with the menstrual cycle or anesthetic management at younger ages. The patient was a 22‐year‐old woman who had developed ventricular fibrillation due to coronary spasm, resulting in cardiopulmonary arrest. She was resuscitated, and underwent ICD implantation. As her symptoms appeared at specific times during her menstrual cycle, she was diagnosed as having menstrual‐associated coronary spasm, and started taking estrogen/progesterone medication. An endometrial ablation was scheduled for endometrial hyperplasia that was caused by the medicine. The surgery was scheduled in consideration of the patient's menstrual cycle, and general anesthesia was selected as the method of anesthesia. The surgery and perioperative management were uneventful, and her postoperative course was favorable. Our case is the first to our knowledge of general anesthesia performed on a patient with menstrual‐associated coronary spasm. To our knowledge, there have been no reports to date of anesthesia management of patients with menstrual‐associated coronary spasm. We encountered a case of a patient with a history of coronary spasm induced by hormonal changes in the menstrual cycle, who was treated with intraoperative prophylaxis, and appropriate anesthesia to prevent coronary spasm attacks during surgery for endometrial ablation. We here report a review of the anesthetic management of this rare case.22). She was scheduled to undergo endometrial ablation for endometrial hyperplasia. Regarding her medical history, she experienced a cardiopulmonary arrest owing to ventricular fibrillation at the age of 15, at which time she was diagnosed as having coronary spasm via a coronary angiogram and acetylcholine stress test. She underwent ICD implantation, together with the initiation of treatment with a Ca2+ blocker and coronary dilators. The ICD was activated four times in 6 years, and the patient resumed a self‐paced heartbeat. After a careful interview regarding the circumstances of the onset of her symptoms, we strongly suspected menstrual‐associated coronary spasm, as her symptoms occurred from just before menstruation to the middle of menstruation. Estrogen/progesterone replacement therapy was started, and her ICD activation and angina attacks ceased. When she was 22 years old, we decided to perform an endometrial ablation with tissue biopsy for endometrial hyperplasia that was thought to be caused by the estrogen/progesterone medication. Her family history included coronary angina pectoris in the father. Her oral medications were diltiazem (120 mg/day), nifedipine (40 mg/day), nicorandil (25 mg/day), and isosorbide mononitrate (40 mg/day), and an isosorbide mononitrate patch (80 mg/day). She had no history of smoking. Her first menstrual period was at 11 years old, and her menstrual cycle was 30 days and regular. Her last menstrual period was 14 days before the scheduled surgery. Blood test findings were normal. Chest X‐ray displayed no cardiac enlargement or abnormal shadows, except for the presence of an ICD device. Preoperative estradiol levels were 63.1 pg/mL in the follicular phase, and 42.9 pg/mL in the ovulatory phase, which were within the normal ranges for these phases in nonpregnant women. Her ECG results were normal, with a pulse of 55 beats/min, sinus rhythm, and a corrected QTc of 403 ms. Transthoracic echocardiography displayed an ejection fraction of 65%, no abnormal wall motion, no systolic or diastolic dysfunction, and no apparent valvular disease. There were load‐induced right ventricular abnormalities, and she had a NYHA classification of grade I. Anesthesia was induced with remifentanil (0.3 μg/kg/min) and propofol TCI (4.0 μg/mL), and muscle relaxation was achieved with rocuronium bromide (50 mg). After endotracheal intubation using a McGrath MAC® video laryngoscope , anesthesia was maintained with propofol TCI (4.0 μg/mL), continuous infusion of remifentanil (0.1–0.2 μg/kg/min), and rocuronium bromide 50 mg, and sedation levels were monitored using the patient state index measured with a Sedline® monitor (Masimo Co.), and maintained at scores between 30 and 50. In addition, an observation arterial pressure line was taken. Intraoperative and postoperative nicorandil (1 μg/kg/min) and diltiazem (1 μg/kg/min) were continuously administered intravenously. During surgery, the ICD was deactivated and a percutaneous external defibrillator was used.The patient was a 22‐year‐old woman  and progesterone (Premarin®) was started. Sugiura et al. reported that oral contraceptives and low‐dose estrogen/progesterone products, regardless of their type, are associated with a significant increase in deep vein thrombosis.There have been no reports on the anesthesia of patients with menstrual‐associated coronary angina pectoris. The present patient required an endometrial ablation, and the attending physician, cardiologist, and anesthesiologist discussed whether the procedure should be performed under general anesthesia or spinal anesthesia..Anesthesia management with spinal anesthesia was initially considered, but there were concerns regarding side effects from the use of various drugs, and the risk of suppression of cardiac function. However, there have been many previous reports of coronary spasms induced by relative parasympathetic dominance due to sympathetic blockade by epidural or spinal anesthesiaAYA KAWACHI: Writing – original draft; writing – review and editing. Saho Sudo: Writing – review and editing. Yusuke Ishida: Writing – review and editing. Koichi Nakazawa: Writing – review and editing.None.The authors declare that they have no competing interests associated with this manuscript.Not applicable.Written informed consent was obtained from the patient for publication of this case report and the accompanying images.