Drug‐associated delirium is a common but potentially preventable neuropsychiatric syndrome associated with detrimental outcomes. Empirical evidence for delirium‐associated medication is uncertain due to a lack of high‐quality studies. We aimed to further investigate the body of evidence for drugs suspected to trigger delirium.https://doi.org.10.17605/OSF.IO/4PUHY).A systematic update review and meta‐analyses of prospective studies presenting drug associations with incident delirium in adult study populations was conducted. Two authors independently searched MEDLINE, PsycINFO, Embase, and Google Scholar dated from October 1, 2009 to June 23, 2020, after screening a previous review published in 2011. The most reliable results on drug‐delirium associations were pooled in meta‐analyses using the random‐effects model. Quality of evidence was assessed using the GRADE‐approach. This study is preregistered with OSF , Olanzapine , Ketamine  or corticosteroids . Low‐level evidence suggests a three‐fold increased risk for anticholinergics . Opioids, benzodiazepines, HWe investigated the retrievable body of evidence for delirium‐associated medication. The results of this systematic review were then interpreted in conjunction with other evidence‐based works and guidelines providing conclusions for clinical decision‐making. Haloperidol, olanzapine, corticosteroids, and ketamine do not exhibit increased risk of delirium .There is insufficient evidence for the use of antipsychotics, corticosteroids, or ketamine as pharmacological prevention or treatment.1‐antihistamines , and polypharmacy might trigger delirium, but underlying evidence remains low.Anticholinergics, opioids , benzodiazepines, HMeta‐analysis results deriving from observational studies do not reach reliable evidence levels. Single study results should be interpreted with caution.Methodological differences in the studies included  limited the certainty of evidence.1Delirium is a serious and common neuropsychiatric disorder that is predominantly reported in elderly patients.1‐antihistamines) is inconsistent and conflicting.Narrative reviews suggest that benzodiazepines, (tricyclic) antidepressants, opioids, and anticholinergics are suspected to have deliriogenic potential.The aim of this systematic review is to update and comprehensively assess the certainty of evidence for drug‐delirium associations, and to conduct meta‐analyses for delirium with specific medication (classes). A systematic literature search is conducted to update a previous review from 2011.2This work is based upon the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions2.1The systematic search required RCTs and prospective observational studies (OS) (prospective cohort [PC] and nested case–control [CC]) with predefined eligibility criteria assessing medication associations with incident delirium in adult study populations. Retrospective study designs, case‐reports, and studies that failed to present exploratory statistics were considered to provide a very low level of evidence, and therefore excluded.2.2A search strategy using Medical Subject Headings (MeSH) and free text search terms was developed in order to systematically screen the MEDLINE (via PubMed), EMBASE, and PsycInfo databases. A complementary search on Google Scholar and screening of reference lists was conducted to extend the scope. The search strategy combined three search strings, reflecting (i) delirium as the dependent variable, (ii) medication exposure as the independent variable, and (iii) focus on elderly people (> 65 years old), by using the Boolean operator “AND.” Eligibility criteria required adult study populations (exclusion of <18 years old patients) with a focus on older patients. English‐, German‐ and French‐language journal articles were included if published between October 1, 2009–to update the previous systematic reviewThe literature search was initially conducted by screening the review by Clegg and Young2.3Data collection of included articles was undertaken by RM while DBJ checked for the accuracy of extracted data. We focused especially on delirium assessment, medication exposure, and statistical approach to ascertain reliable data and assess the possible risk of bias. Adjusted and unadjusted effect measures, as well as raw data, were collected.2.4age was defined as a primary confounder, and dementia and comorbidity/illness severity as joint secondary confounders. A score of ≥7 was regarded as indicative of sufficient bias control. There was a risk of bias, if more than one point in a single category was subtracted.RCTs were appraised using the Cochrane risk of bias tool (RoB).2.5Meta‐analyses were conducted if there were at least two studies investigating the same medication. These were pooled using the random‐effects model.p < 0.05) and the I2 statistic.Pooled odds ratios (ORs) with 95% confidence intervals (CI) were calculated for dichotomous outcomes (Cochran–Mantel–Haenszel test or generic inverse‐variance method). Statistical heterogeneity was calculated using the χ2 test . In full text screening, the eligibility criteria ,The RCTs included were based upon intention‐to‐treat (ITT) analyses with the following exception: two RCTs conducted per‐protocol (PP) analyses,3.3The RoB among the RCTs included was estimated to be low, and is presented in Figure 3.4Single medication associations (uni‐ and multivariable effect estimates and raw data) are presented in a comprehensive summary table in the Appendix 3.5We established a hierarchy of evidence, including meta‐analyses and single effect estimates on medication associations with delirium. Comprehensive meta‐analyses and sensitivity analyses are presented in the Appendix 3.5.12 of 44% (p = 0.13).There is high quality evidence from five pooled RCTsmoderate) due to insufficient optimal information size (OIS).The moderate quality evidence of one RCTmoderate because of insufficient OIS. Heterogeneity calculations showed a borderline result  but sensitivity analyses did not change results significantly . Borderline heterogeneity might be explained by different agents and dosages  was accepted because point estimates did not differ in direction. The GRADE‐rating had to be downgraded by one level to moderate due to serious imprecision.There is moderate quality of evidence from two RCTslow) due to very serious imprecision. Atropine (0.01 mg/kg i.m.) shows an insignificant result . PHC shows a strong association with delirium, but confidence intervals are very broad .There is low quality evidence from one RCT3.5.2very low quality of evidence: ACE‐inhibitors, antibiotics, oral anticoagulants, antidepressants, antiparkinsonian drugs, benzodiazepines, beta‐blockers, calcium‐channel blockers, digoxin, diuretics, H1‐ and H2‐antihistamines, heparin, Insulin, NSAIDs, paracetamol, opioids, propofol and polypharmacy (as a delirium‐associated drug entity). The results for selected medication that is commonly regarded as being delirium‐associatedAll other medication classes investigated in OS reached only very low due to serious imprecision.Antidepressants as a general drug class were investigated in one study (89 patients with negative dementia status undergoing spine surgery).very low due to serious inconsistency (I2 = 76%) and serious imprecision. AR estimates could not be calculated. Sensitivity analysis .Benzodiazepines did not significantly increase or decrease delirium rates in our meta‐analysis of four PCsvery low level because upgrading (dose–response gradient) was not possible due to prior downgrading (serious inconsistency and imprecision).A dose–response gradient is shown in four OSvery low due to serious imprecision.Among specific agents, lorazepam exhibited an increased risk for delirium and a dose–response gradient in 198 ICU‐patients (aOR of 1.24 [95% CI: 1.08–1.43]).2 of 85% which might be explained by heterogeneous clinical settings : aOR of 0.75, 95% CI: 0.14–4.16. AR resulted in 19 fewer cases per 1000 (95% CI: 70 fewer to 189 more). Evidence quality needed to be downgraded to very low due to serious inconsistency (I2 = 83%) and imprecision. Heterogeneity might be explained by the fact that H1‐antihistamines as a medication classMeta‐analysis of two CCs2 = 0%, p = 0.38).1‐antihistamines as a medication class (very low) because the same studies were included.Heterogeneity was low when results for diphenhydramine were pooled (I2 of 47% (p = 0.15). Confidence in evidence needed to be downgraded to very low due to serious imprecision. AR was not calculable due to a lack of raw data in one PC.Meta‐analysis of three PCsOpioids exhibited a dose–response gradient: a postoperative dose of >8 mg hydromorphone‐equivalent in comparison to <8 mg results in an adjusted OR of 2.60 (95% CI: 1.31–5.17)very low : the meta‐analysis .The specific agents morphine, fentanyl and meperidine showed higher possible risk than other opioid agents, although evidence levels are 2 = 0%). AR estimates could not be calculated. The GRADE‐rating for fentanyl and morphine was downgraded to very low due to serious imprecision.The same two studiesOne PCvery low due to insufficient OIS.In comparison, oxycodone and codeine did not show an association with delirium .2 = 67%), possibly due to heterogeneous definitions of polypharmacy  remains incomplete, and mostly of low quality, but some findings deserve attention, especially in the context of other evidence‐based works.The NICE‐guidelinesCholinergic deficiency and anticholinergic toxicity are considered the main pathophysiological correlates in delirium genesis.Only one PC investigated antidepressants as a group and presented an adjusted (insignificant) association with very low‐evidence certainity.not a significant risk factor.”Benzodiazepines are described as major pharmacological risk factors for inducing delirium due to paradoxical effects.1‐antihistamines  are usually considered deliriogenic.1‐antihistamines of “low to moderate quality.”1‐antihistamines as medication class). The NICE guidelines present “low quality evidence” for an increased risk from diphenhydramine and “very low evidence” not significantly altering delirium risk.1‐antihistamines in general and diphenhydramine in special), but relatively higher evidence suggests increased delirium risk.The anticholinergic properties of first generation Hnot to alter risk of delirium occurrence” in ICU settings.Opioid exposure is considered a well‐established independent risk factor for delirium, although empirical evidence (from OS) is inconsistent.Polypharmacy–especially the use of multiple psychoactive drugs ‐ was described as a major delirium risk factor  by Inouye et al.single triggers for delirium : ACE‐inhibitors; antibiotics, oral anticoagulants, antiparkinsonian drugs, beta‐blockers, calcium‐channel blockers, digoxin, diuretics , H2‐antihistamines, heparin, Insulin, NSAIDs, paracetamol and propofol. Notably, Clegg and Young describe dihydropyridines (nifedipine) as showing a “small to moderate” risk ,The GRADE quality of evidence ratings for all other investigated medication classes and agents were of a very low level. The following are therefore not regarded as playing a major role as 4.1This systematic review on delirium‐associated medication is the first work pooling individual studies and providing meta‐analyses according to medication classes and individual agents. Another strength lies in comprehensively grading the quality of evidence in a structured, transparent and standardized manner. We also compared our results to previous reviews and delirium guidelines.Although focusing on multivariable adjusted effect measures, there are limitations to evidence levels due to heterogeneous clinical settings, broad ranges of sample sizes and delirium rates, and lack of à priori power calculations, frequently resulting in insufficient OIS and imprecision estimation. The accounted covariates in multivariable regression analyses were inconsistent across studies and might affect comparability. The number of pooled studies included in meta‐analyses was too low to conduct funnel‐plots and estimate publication biases. Negative results were not reported systematically across the studies included.4.2This critical appraisal of current evidence on delirium‐associated medication has both clinical and research implications.Haloperidol (up to 6 mg/d) and olanzapine (10 mg) do not increase the risk of delirium . Nevertheless, there is insufficient evidence for the use of antipsychotics as pharmacologic prevention or treatment.Corticosteroids and ketamine (in subanesthetic dosage: up to 1 mg/kg) did not exhibit significantly increased delirium risk .Anticholinergics–whenever possible–should be de‐prescribed or avoided. There is low quality evidence for a three‐fold increased delirium risk.Opioids show a dose‐dependent increased delirium risk , and insufficiently treated acute pain seems to be a strong precipitating factor. We suggest avoiding meperidine. Oxycodone seems to have a favorable profile. Opioids do not seem to be a risk factor in the ICU‐setting.Exposure to benzodiazepines can dose‐dependently increase the risk of delirium, but evidence is of very low quality in non‐ICU‐settings. In ICU‐settings, there is strong evidence for increased risk . Lorazepam exhibited a higher risk compared to other agents , and showed a strong dose‐effect relationship, but quality of evidence is too low to draw clinical conclusions. We therefore suggest using non‐benzodiazepine sedatives 1‐antihistamines–especially diphenhydramine–seem to increase delirium risk, but evidence remains inconsistent and of very low quality.HPolypharmacy might strongly contribute to delirium but there is a lack of empirical evidence.continuous delirium assessment and adequate follow‐up time, sufficient statistical power calculations, predefined confounders , standardized dosages, and the sub‐categorization of delirium in the context of suspected drug‐induced pathophysiology .We recommend prospective study designs for future research, with Although the overall evidence is of low quality we suggest the following:51‐antihistamines, antidepressants) did not reach a reliable quality for drawing conclusions. Nevertheless, a synopsis including other evidence‐based works and guidelines shows that benzodiazepines are a strong dose‐dependent risk factor in the ICU. In non‐ICU‐settings, however, the evidence remains unclear. Opioids  can trigger delirium in a dose‐dependent manner, but insufficient analgesia in acute pain is a precipitating risk factor per se. In ICU‐settings, opioids do not seem to significantly contribute to delirium. H1‐antihistamines (diphenhydramine) seem to increase delirium risk although the evidence is very low. Polypharmacy might be a major condition, but empirical evidence is unclear. Medication reviews, avoiding and de‐prescribing possibly deliriogenic medication in people at risk is crucial in clinical practice. This review may help clinicians to prevent drug‐induced delirium.Meta‐analyses and GRADE certainty of evidence confirm the paucity of high‐quality studies. In summary, antipsychotics, corticosteroids and ketamine (in subanesthetic dosage) did not demonstrate increased delirium risk with moderate to high certainty evidence. Low quality evidence suggests a three‐fold increased risk with primary anticholinergics. Medication classes investigated in OS (e.g., opioids, benzodiazepines, HMichael Reisinger, Daniela Schoberer, and Eva Z. Reininghaus formulated the study design. Michael Reisinger pre‐registered the study protocol. Michael Reisinger and Johanna De Biasi conducted literature searches independently. Daniela Schoberer was consulted in case of disagreement on eligibility. Michael Reisinger extracted data, Johanna De Biasi checked for accuracy. Michael Reisinger and Daniela Schoberer contributed to data analysis, meta‐analyses and interpretation. Michael Reisinger wrote the first draft of the manuscript and prepared figures and tables. Daniela Schoberer, Eva Z. Reininghaus and Frederike T. Fellendorf edited the manuscript.This study did not receive financial or nonfinancial support from any funding agency in the public, commercial, or nonprofit sectors.We declare no competing interests.https://publons.com/publon/10.1111/acps.13505.The peer review history for this article is available at Appendix S1 Supporting Information.Click here for additional data file.Appendix S2 Supporting Information.Click here for additional data file.Appendix S3 Supporting Information.Click here for additional data file.Appendix S4 Supporting Information.Click here for additional data file.


To evaluate the analgesic efficacy and safety of different does of intravenous ibuprofen (IVIB) in the treatment of postoperative acute pain.  Patients with an intravenous (IV) patient-controlled analgesia device after abdominal or orthopedic surgery were randomly divided into placebo, IVIB 400 mg, and IVIB 800 mg groups. The first dosage of study medicines was given intravenously 30 minutes (min) before surgery ended, followed by six hours (h) intervals for a total of eight doses following surgery. The demographic characteristics and procedure data, cumulative morphine consumption, the visual analog scale (VAS), the area under the curve (AUC) of VAS, patient satisfaction score (PSS), the rates of treatment failure (RTF), and adverse events (AEs) and serious adverse event (SAEs) were recorded during the period of trial. P = 0.0011) and the IVIB 800 mg group  was significantly reduced compared with the placebo group (14.51 ± 9.19 mg) for 24 h postoperatively, there was no significant difference between the IVIB 400 mg and IVIB 800 mg groups (P = 0.9997). The placebo group had significantly higher VAS and the AUCs of VAS than those in the IVIB 400 mg and the IVIB 800 mg groups at rest and movement for 24 h postoperatively (P < 0.05), and there was no significant difference between the IVIB 400 mg and IVIB 800 mg groups (P > 0.05). RTF was slightly higher in the placebo group than IVIB 400 mg group and 800 mg group, and no statistical significance (P < 0.690). PSS in the IVIB 400 mg (P = 0.0092) and the IVIB 800 mg groups (P = 0.0011) was higher than the placebo group for pain management, there was also no significant difference between the IVIB 400 mg and IVIB 800 mg groups (P = 0.456). The incidence of RTF (P = 0.690) and AEs (P > 0.05) were not different among the three groups.  A total of 345 patients were enrolled in the full analysis set (FAS), and of 326 participants were valid data set (VDS). Demographic characteristics, disease features, and medical history of patients were not significantly different between groups. Total morphine consumption of the IVIB 400 mg group (11.14 ± 7.14 mg;  Intermittent IV administration of ibuprofen 400 mg or 800 mg within 24 h after surgery in patients undergoing abdominal and orthopedic surgery significantly decreased morphine consumption and relieved pain, without increasing the incidence of AEs. Postoperative pain has long been a major concern for patients. A Spanish multicenter cross-sectional survey found that 73% of patients experienced acute postoperative pain . About 6μ, κ, and δ receptors, which is not the most effective against pain caused by nerve injury and inflammation, and one of the reasons for the failure of postoperative acute pain management [Opioids are widely used in acute pain treatment because of their powerful analgesic effect. Early studies have shown that more than half of patients received morphine to relieve moderate or severe postoperative pain , 10. Hownagement , 12. In nagement . The Amenagement  that a mnagement –16. ClinThere are various NSAIDs in clinical, but they may increase the risk of gastrointestinal bleeding with limited onset of action and repeated doses . IbuprofConcerning adverse events (AEs), a study confirmed that ibuprofen is less prone to gastrointestinal (GI) and cardiovascular than ketorolac and diclofenac , and theThe ethics of the study has been approved. Drug clinical research approval number: 2014L01894; date of registration: 16/01/2015. All methods were performed by the relevant guidelines and regulations. Informed consent forms and all amendments were reviewed and approved .This study was a prospective, randomized, double-blind, placebo-controlled trial. This study was conducted in 10 hospitals throughout China from Jul.10, 2015, to Dec.25, 2016.A total of 360 patients under general anesthesia were included in this study, all of whom were abdominal and orthopedic surgery patients with incisions length 5 cm at least and who required postoperative hospitalization . Pain in2 and/or BMI ≥ 30 kg/m2, a history of asthma or heart failure, or pregnant or nursing or poorly controlled hypertension  were also excluded. Patients were excluded from the study if they had a history of allergy or hypersensitivity to NSAIDs or used NSAIDs within 12 h, or had a history of tolerance or dependence on narcotics or opioids. Patients with kidney dysfunction  or liver dysfunction (ALT or/and AST was greater than 1.5 times the limit of the baseline) were unable to participate in this study. Patients were considered when they had no underlying bleeding factors such as hemophilia, thrombocytopenia, abnormal platelet function; or no used other analgesics, muscle relaxants, or sedatives within 24 h prior to surgery; or no local anesthesia, nerve blocks, epidurals, and another analgesia preoperatively/intraoperatively. Patients were excluded if taking warfarin, lithium, combined angiotensin-converting enzyme inhibitors (ACEI), and diuretics at present; or with other events deemed by the investigator that were not eligible, for example, enrolled in other clinical studies within 3 months . The trial medication and prophylactic antiemetics ) was given at half an hour before the end of the operation. There were no other catheter-based regional anesthetic techniques Figure 4The method of centralized random grouping was adopted, and each research center competed to be included in the group. During the first 48 h of the study period subjects were randomized in a 1 : 1 : 1 ratio to the IVIB 400 mg group (diluted to 200 ml 0.9% NaCl), the IVIB 800 mg group (diluted to 200 ml 0.9% NaCl), and the placebo group (200 ml 0.9% NaCl) using a double-blind, simple randomization protocol. The first dose of ibuprofen or placebo was administered 30 minutes before the end of the procedure. Subsequently, interventions were performed every 6 h until the end of the whole clinical trial, with a total of 8 doses . All patResearchers, patients, and caregivers were unaware of the intervention allocation, and only supervising physicians were known.(1) Primary Outcome. Total morphine use in 24 hours after surgery.(2) Secondary Outcomes. ① Visual analogue scale (VAS) was measured by patient self-assessment both at rest and with movement . Different surgical types of postoperative pain with movement may bring on or aggravated by differently movement. In this paper, cough, physiotherapy, sitting, standing, rolling over in bed, and active flexion and extension were specifically described as pain-evoking maneuvers. ② The area under the curve (AUC) of VAS  at rest and with movement; ③ the rates of treatment failure (RTF), defined as the rate of analgesia with other nonmorphine drugs within 24 h after the end of surgery; ④ patient satisfaction score  was recorded on postoperative day 2.Specific AEs associated with IVIB include renal dysfunction, cardiovascular events, GI or operative site bleeding, thrombophlebitis, gastrointestinal dysfunction , and nervous system (headache and dizziness). Opioid-related specific AEs include nausea, vomiting, pruritus, respiratory depression, sedation, urinary retention, and allergic reactions/rash .α = 0.025, 1-β = 80%. The data showed a difference of 8.6 mg in total dosage of morphine between the IVIB 800 mg ibuprofen group and the placebo group 24 h after surgery, with a combined variance of 400 mg [To hypothesis that the effect of the 800 mg ibuprofen group was better than the placebo group, and the study design was 1 : 1, when the main efficacy point was the total dose of morphine 24 h after surgery. The parameters were set: bilateral f 400 mg . Accordit-tests for the data before and after treatment within each group, if the variable followed normal distribution. AEs, SAEs, and RTF were tested by the χ2 test or fisher's exact to compare differences between groups. Medical history and PSS was analyzed using a Kruskal–Wallis H test. P < 0.05 was considered statistically significant (two-sided test). All statistics were analyzed in SAS 9.4 software (SAS Institute Inc.).Percentages present categorical variables. Interquartile range presents level variable. If the continuous variables conformed to a normal distribution, parametric tests, such as one-way analysis of variance (ANOVA), were used, and the results were expressed as means ± standard deviation (SD); if they did not conform to a normal distribution, nonparametric tests, such as rank sum tests, were used, and the results were expressed as median and interquartile range. Therefore, ANOVA and the LSD test were used to compare different groups' continuous variables . Laboratory assessments were analyzed using paired A total of 345 patients were enrolled to full analysis set . Of these, 326 populations were enrolled to valid data set  . BaselinP = 0.0011) and the IVIB 800 mg group  morphine consumption was significantly reduced compared with the placebo group .IVIB 400 mg group , was significantly reduced at 1 h , 3 h , 6 h , 12 h , 24 h , 36 h , and 48 h  after surgery over time , 6 h , 12 h , 24 h , 36 h , 48 h  after surgery, compared with the placebo group (P < 0.0001) . PSS was higher in the IVIB 400 mg (P = 0.0092) and the IVIB 800 mg (P = 0.0011) group than in the placebo group, but not significantly different from the IVIB 400 mg and IVIB 800 mg groups . Supplemental instrument P = 0.0002). Furthermore, the placebo group had a significantly higher body temperature than the IVIB group, from 6 h after the administration of the first dose to the end of the study , IVIB 400 mg group of 115 patients (60.36% and 1.74%), and IVIB 800 mg group of 111 patients (68.91% and 2.70%), respectively, and there were no differences between groups  enzyme . A recenThe strength of this study was that it is a multicenter, randomized, controlled, and prospective study. Not only were dynamic pain intensity scores considered as the important concern, but also the AUC of VAS for each time period and patient satisfaction were included in the outcome index, which was essential for the recovery of overall function. More importantly, we indicated the lack data of treatment for IVIB 400 mg on postoperative pain, compared to 800 mg and added the existing data to explain that 30 min infusion time and q6h of IVIB 400 mg and 800 mg potentially reduced complications associated with postoperative fever, such as longer hospitalization, avoidable healthcare costs, and increased morbidity for patients. In a word, it provided physicians with strategies for ibuprofen-assisted postoperative analgesia by assessing the safety and efficacy.The disadvantage was that this protocol failed to extrapolate tolerability results including relatively unhealthy patients that underwent elective surgery through broadening the inclusion/exclusion criteria. For SAEs, more detailed documentation and analysis should be done. Further, we look forward to further studies of similar design to validate the analgesic effect of intravenous ibuprofen for postoperative acute pain to add scientific validity to the conclusions, especially with IVIB 400 mg.In conclusion, the results of this study suggest that IVIB 400 mg and 800 mg ibuprofen administrating postoperative pain before 30 minutes the end of surgery, with Injection every 6 hours, 8 times in total, can reduce morphine consumption, which may reduce recognized opioid-related AEs associated with postoperative analgesia, also achieved effective pain relief. Regarding the incidence of AEs associated with IVIB, gastrointestinal dysfunction, bleeding, nephrotoxicity, and cardiovascular or neurological events, there were no significant differences between the 400 mg group, the 800 mg group, and the placebo group; IVIB was well tolerated for postoperative pain treatment.


Numerous studies have shown that the insulin‐like growth factor (IGF) pathway is highly associated with tumor initial and progression in several tumors. However, compared with IGF1/1R and IGF2/2R, insufficient studies have focused on IGF‐binding proteins (IGFBPs).The GDC TCGA and GTEx data of 33 cancers, TCGA pan‐cancer immune phenotypes, tumor mutation burdens, and the copy number alterations of IGFBPs were extracted. Next, the prognostic value of IGFBPs was analyzed based on a univariate Cox analysis. Additionally, the ESTIMATE algorithm was used to calculate stromal and immune scores and tumor purity, and the CIBERSORT algorithm was used to estimate tumor‐infiltrating immunocyte levels. Ultimately, the correlation between IGFBP expression and cancer hallmark pathways was estimated with a Spearman analysis.The expression of IGFBPs was differentially expressed and correlated with prognosis in specific cancers. IGFBPs may operate as biological markers for carcinogenesis and progression and as prognostic biomarkers. Additionally, IGFBP5 has been proved that promotes the invasion and migration of ovarian cancer.In general, IGFBPs can serve as predictable biomarkers and potential therapeutic targets for specific tumors. Our results could provide underlying targets for the design of laboratory experiments to elucidate the mechanism of IGFBPs in cancers and identify IGFBP5 as a prognostic factor in ovarian cancers. This system includes three major ligands , cell surface receptors , at least six circulating IGF‐binding proteins (IGFBPs), and ten IGFBP‐related proteases (IGFBP‐rPs).In this article, we focused on IGFBP mRNA across cancers. First, the expression of IGFBPs was studied, and the effect of IGFBPs on tumor prognosis was evaluated. Next, we investigated the genetic alteration of IGFBPs. Additionally, the association between IGFBPs and three hallmarks of cancer, evading immune destruction, activating invasion and metastasis, and reprogramming energy metabolism was analyzed. Ultimately, we verified the expression and prognostic effect of IGFBPs in ovarian cancer, and the biologically relevant pathways in ovarian cancer were analyzed. Our results revealed that IGFBPs may be used as prognostic factors for a variety of cancers and play important roles in tumor invasion and metastasis and reprogramming energy metabolism. Validation results in ovarian cancer suggested that IGFBP5 can be a candidate and promising prognostic marker.22.1https://xenabrowser.net/datapages/). Although the TCGA project also included the sequencing data of adjacent normal tissue samples from some patients, these “normal” tissue samples may be affected by neighboring tumor cells, which is why GETx samples were applied to match normal tissue.https://www.ncbi.nlm.nih.gov/geo/). Ultimately, the 33 cancers are adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma (GBM), brain lower grade glioma (LGG), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), mesothelioma (MESO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS), and uveal melanoma (UVM).Based on the Cancer Genome Atlas (TCGA) and the Genotype‐Tissue Expression (GTEx) data of 33 cancers, the clinical data of overall survival (OS), disease‐free interval (DFI), progression‐free interval (PFI), disease‐specific survival (DSS) for each TCGA cancer type and TCGA pan‐cancer immune phenotype data were downloaded from the USCS Xena browser (2.2http://timer.comp‐genomics.org/).https://kmplt.com/analysis/) consisting of GEO, the European Genome‐phenome Archive , and TCGA was employed to validate the relationship between IGFBPs and the OS of patients affected by 21 distinct types of cancer.A univariate Cox proportional risk regression model was used to test the association between IGFBP gene expression and OS, DFI, PFI, and DSS in 33 cancers. Based on the mean expression of IGFBPs, patients were divided into high or low groups in various tumors. In addition, the clinical relevance of IGFBPs across various cancer types was obtained from TIMER2.0 .2.4https://www.gsea‐msigdb.org/gsea/msigdb/index.jsp). Furthermore, we acquired four previously reported metabolic signature gene sets, including amino acid metabolism, carbohydrate metabolism, lipid metabolism, and tricarboxylic acid cycle (TCA).Four tumor‐related biological pathways  were downloaded from the molecular signature database (MsigDB) .http://ualcan.path.uab.edu/analysis‐prot.html).All patients provided informed consent and were approved by the Ethics Committee of the Renmin Hospital at Wuhan University. Normal ovarian tissue was collected from four patients with benign ovarian tumor. We also obtained ovarian cancer tissue from six patients, and paracancer tissue from two of these patients. The clinical parameters were displayed in Table 2.6SKOV3 cells are human serous OC cell lines that were obtained from the American Type Culture Collection (ATCC) cell bank. And the SKOV3 cells were cultured as in our previous results.2.7SKOV3 without or with IGFBP5 recombinant protein  were cultivated in 200 μL of medium without FBS and then placed in the upper chamber with Matrigel‐coated membrane . Next, 600 μL of 1640 medium containing 10% FBS was introduced into the bottom chamber. After incubation for 24 h, the cells were fixed and stained with crystal violet.2.8SKOV3 cells without or with IGFBP5 recombinant protein were seeded onto 6‐well plates. When the cells covered the entire bottom of the 6‐well plates, the 200 μL pipette tip was used to scratch the adherent cell layer. Subsequently, cells were incubated in an FBS‐free medium for 24 h. Wound healing capacity was monitored via microscopy at 0, and 24 h.2.9Total protein was extracted from cells using protein lysate, which consisted of RIPA buffer (Bioss), PMSF (Beyotime Biotechnology), total protease inhibitor (Servicebio), and phosphatase inhibitor (Servicebio). Next, protein lysates were loaded onto 10% or 12.5% SDS‐polyacrylamide gels (Yeasen Biotechnology Co. Ltd.). Next, protein lysates were transferred to 0.45 mm/0.22 mm polyvinylidene difluoride (Merck Millipore) for 60 min. The membranes were blocked for 1 h at room temperature with 5% nonfat milk. Then, the membranes were incubated with primary antibodies against IGFBP5 , E‐Cadherin , N‐Cadherin , MMP9 , MMP2 , α‐tubulin  or GAPDH  overnight at 4°C. Subsequently, the membranes were incubated with HRP Goat Anti‐Rabbit IgG solution  or HRP Goat Anti‐Mouse IgG solution  for 1 h at room temperature. After washing three times with TBST for 5 min each time, the membranes were covered with ECL reagents . The intensity of protein bands was quantified by Image J software (Ver 1.5.3).2.10https://www.gsea‐msigdb.org/gsea/msigdb/index.jsp). The top 10 terms of HALLMARK analyses are shown. Gene sets with p < 0.05 were considered significantly enriched.GSEA was conducted to investigate the biological functions in the high‐ and low‐risk groups of each IGFBP with the cut‐off value of IGFBPs. HALLMARK gene sets were downloaded from the MSigDB database  was employed to conduct a source analysis. Using the “limma” R package, gene expression was compared between normal tissue and neoplasms from 25 cancer types with more than five adjacent normal samples or normal tissue samples. Gene expression data from the TCGA and GTEx databases were analyzed using Student's 33.1p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure We first matched each solid cancer type in TCGA with the corresponding healthy tissue from the same tissue source in the GTEx project, such as normal ovarian tissue from GTEx and ovarian cancer from TCGA. After the integration of GTEx and TCGA data, the expression levels of IGFBPs in 33 types of tumors were analyzed. The relevant information on the 33 types of cancers is shown in Table 3.2p < 0.05). IGFBP2 played a detrimental role in LGG, UVM, KIRP, GBM, UCEC, and KIRC. IGFBP3 was a high‐risk gene in LGG, MESO, KIRP, THYM, READ, PAAD, LIHC, and LUAD (p < 0.05). In addition, the increased expression of IGFBP4 predicted worse OS in LGG, GBM, UVM, OV, and MESO and better OS in LIHC, UCEC, BRCA, and KIRP (p < 0.05). IGFBP5 had a detrimental effect on LGG, UCEC, KIRP, STAD, and BLCA (p < 0.05). IGFBP6 is a low‐risk gene in KIRP, BRCA, and PRAD. By contrast, it is a high‐risk gene in GBM, KICH, ACC, LGG, and STAD (p < 0.05). Furthermore, the results of the TIMER 2.0 database were in line with the Cox proportional hazards model. The results revealed that high levels of IGFBP1 were associated with a poor prognosis in ESCA, HNSC, KIRC, LUAD, SARC, STAD, and THYM (p < 0.05). IGFBP2 played a detrimental role in KIRP, GBM, UCEC, and UVM but the opposite role in PAAD (p < 0.05). A higher level of IGFBP3 correlated with longer survival times in BRCA and SKCM, but predicted a poor prognosis in KIRP, LGG, LIHC, LUAD, MESO, PAAD, READ, and THYM (p < 0.05). IGFBP4 was a risk gene in GBM, LGG, MESO, OV, SKCM, STAD, and UVM and a protective factor in UCEC, LIHC, KIRP, and BRCA (p < 0.05). The expression of IGFBP5 was linked with poor prognosis in BRCA, KIRP, STAD, and UCEC (p < 0.05). IGFBP6 expression was associated with poor prognosis in ACC, HNSC, KICH, LGG, and STAD (p < 0.05). Additionally, we applied a Kaplan–Meier plotter based on the GEO, EGA, and TCGA databases to validate the results . IGFBP2 correlated with worse survival time in LGG and THCA and longer survival time in THCA (p < 0.05). The expression of IGFBP3 was linked to worse prognosis in PAAD, LGG, and LIHC (p < 0.05). IGFBP4 played a protective role in UCEC (p < 0.05). IGFBP5 played a detrimental role in CESC and PAAD and a protective role in SARC (p < 0.05). IGFBP6 was a risk gene in ACC and PAAD and a protective factor in KIRP (p < 0.05). For DSS and PFI, the results are shown in Tables p < 0.05). Additionally, IGFBP3 was a risk factor in LIHC and IGFBP4 was a protective factor in UCEC (p < 0.05). The expression of IGFBP6 was associated with longer survival time in KIRP among four Cox proportional hazards model analyses (p < 0.05).OS is widely used as an endpoint for cancers because ambiguity in defining this event is minimal; the patients are either alive or dead. However, longer follow‐up is required to obtain OS data for each patient. Therefore, using OS as an endpoint may weaken a clinical study because death due to causes other than cancer does not necessarily reflect tumor biology, aggressiveness, or responsiveness to therapy.3.3p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure Tumorigenesis is a complex multi‐step process involving the interaction of genetic changes.3.4p < 0.05). Interestingly, the correlation between IGFBPs and stromal scores was consistent with the trend of the immune score. The immune and stromal scores reflect the abundances of the immune infiltrate and stromal cells, respectively, and higher respective scores indicate a larger proportion of the corresponding component in the TME. Tumor purity can reflect the ratio of tumor cells component in TME. Regarding tumor purity, the expression of IGFBPs negatively correlated with tumor purity, except for IGFBP2 in PCPG, KIRP, and OV , IFN‐gamma Dominant (Immune C2), Inflammatory (Immune C3), Lymphocyte Depleted (Immune C4), Immunologically Quiet (Immune C5), and TGF‐beta Dominant (Immune C6).t Figure .3.5p < 0.05, Figure p < 0.05, Figure Cancer invasion and metastasis and metabolic reprogramming are two typical hallmarks.p < 0.001, Figure p < 0.001). With regard to carbohydrates, IGF1, IGF2, and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development, and growth of the organism.Accumulated evidence has corroborated that metabolic reprogramming could affect the immune response and clinical prognosis in cancer patients.3.6p < 0.05, Figure We know from the above results that IGFBPs are downregulated in ovarian cancer and IGFBP3/4/5/6 is positively correlated with immune scores in ovarian cancer. In addition, they are closely associated with EMT, angiogenesis, hypoxia, and carbohydrate in ovarian cancer. Therefore, we selected the patients with ovarian cancer for validation. We used PCR to study the mRNA expression level of IGFBPs, which suggested that the expression levels of IGFBPs were downregulated in ovarian cancer. Additionally, we obtained the protein expression levels of IGFBPs in the CPTAC ovarian cancer cohort. Unfortunately, data on the protein expression level of IGFBP1 in ovarian cancer are lacking. In the CPTAC cohort, IGFBP3, 4, 5, and 6 were significantly downregulated in ovarian cancers, consistent with the mRNA expression levels . Specifically, IGFBP1/3/4/5/6 positively regulated EMT (p < 0.05), which was consistent with previous results in pan‐cancer and GSE27612.GSE26712 was used to explore the prognostic role of IGFBPs in ovarian cancer. We performed a univariate Cox and survival analysis based on IGFBPs expression in different ovarian cancer patients and the results revealed that IGFBP3/4/5/6 was the risk factor Figure . After c4In this article, we report the expression of IGFBPs among various cancers and analyzed the prognostic value of IGFBPs. Notably, the expression levels of IGFBPs were downregulated in both ovarian and breast cancer, whereas increased expression levels of IGFBPs were observed in GBM and PAAD. Additionally, among the four Cox proportional hazards model analyses of OS, DFI, PFI, and DSS, IGFBP1 was a risk gene in STAD. In LGG, IGFBP2 and IGFBP3 play a detrimental role. IGFBP4 was a protective factor in UCEC. The expression of IGFBP6 was associated with a longer survival time in KIRP. The results suggested that IGFBPs were differentially expressed in certain cancers and corresponding controls, which also correlated with the prognosis of patients.Genome instability, evading immune destruction, activating invasion and metastasis, and reprogramming energy metabolism were typical hallmarks of tumors.To evade immune destruction, weakly immunogenic cancer cells are believed to be able to grow and generate solid tumors, whereas highly immunogenic cancer cells are eliminated by the immune system.Regarding activating invasion and metastasis, EMT is a process in which epithelial cells acquire mesenchymal morphology and can be activated temporarily or stably to varying degrees during the invasion and metastasis of cancer cells.Metabolic reprogramming is essential for the unlimited reproduction of cancer cells and affects the tumor immune response and clinical prognosis in cancer patients.Finally, we analyzed the IGFBP mRNA expression in ovarian cancer samples, and the results suggested that the expression levels of IGFBPs were downregulated in ovarian cancer. Additionally, the protein expression levels of IGFBPs in the CPTAC cohort were consistent with the mRNA expression levels, except for the expression of IGFBP2. Subsequently, GSE26712 was employed to validate the results of the previous pan‐cancer analysis. The results revealed that IGFBP3/4/5/6 was the risk factor in OC. After correction using multivariate analysis, we observed that IGFBP3 and IGFBP5 remained risk factors. Compared with IGFBP3, the studies on the relationship between IGFBP5 and ovarian cancer are limited. In GSE26712, we divided ovarian cancer patients into two risk groups based on the expression of IGFBP5. Next, we compared the levels of four tumor biological pathways in two risk groups and observed that the EMT and hypoxia pathways were enriched in the high‐risk group, while glycolysis was enriched in the low‐risk group. Additionally, IGFBP5 was proved which promotes the invasion and migration of ovarian cancer cells. Furthermore, we used the GSE140082 dataset for external validation, and the results were consistent with the results of GSE26712 and pan‐cancer.In summary, the pan‐cancer analysis examined the expression of IGFBPs and their prognostic value in various cancers. Next, the associations between four typical hallmarks of tumors and IGFBPs were comprehensively analyzed. Our data suggested that IGFBP5 can serve as an independent prognostic factor in ovarian cancer. However, some limitations remained. First, we matched each solid cancer type in TCGA with the closest corresponding healthy tissue in the GTEx. Because the source of patients and specific information were not clear, this comparison is subject to bias. Second, this study mostly focused on bioinformatic analysis, and some correlations described herein are only suggestive. Therefore, the mechanism by which IGFBPs specifically affect the tumor progression requires additional in vitro and in vivo experiments.5Additional knowledge of the complex regulatory mechanisms of IGFBPs is essential to better exploit its potential clinical utility for diagnostic and therapeutic purposes. This study revealed that IGFBPs are closely correlated with tumor invasion and metastasis. Additionally, IGFBPs may regulate the metabolic reprogramming to mediate tumor carcinogenesis. The PI3K and MAPK signaling pathways may be involved in the biology by which IGFBPs mediate tumor invasion, migration, and carbohydrate production. Our results could provide underlying targets for the design of laboratory experiments to elucidate the mechanism of IGFBPs in cancers and identify IGFBP5 as a prognostic factor in ovarian cancers. Further studies focusing on the mechanisms of IGFBPs at both the cellular and molecular levels, especially investigating the mechanisms underlying positive correlations, will be beneficial to clarify the role of IGFBPs among cancers.Wei Tan: Conceptualization (lead); writing – original draft (lead). Jie Zhang: Investigation ; resources . Zhimin Deng: Methodology ; validation . Fangfang Dai: Data curation ; visualization . Lujia Tang: Formal analysis ; software . Wei Hu: Funding acquisition ; writing – review and editing . Hua Liu: Funding acquisition (lead); writing – review and editing .This work was supported by cross‐innovation talent project in Renmin Hospital of Wuhan University (Grant No. JCRCZN‐2022‐016); the Natural Science Foundation of Hubei Province (Grant No. 2022CFB252); the Undergraduate education quality construction comprehensive reform project (Grant No. 2022ZG282); and Innovative Research Group Project of the National Natural Science Foundation of China (Grant No. 82071655).The authors declare no competing interests.This study was approved by the Ethics Committee of the Renmin Hospital of Wuhan University and carried out following the Declaration of Helsinki.Figure S1.Click here for additional data file.Table S1.Click here for additional data file.


Isolated remethylation defects are rare inherited diseases caused by a defective remethylation of homocysteine to methionine, preventing various essential methylation reactions to occur. Patients present with a systemic phenotype, which can especially affect the central and peripheral nervous systems leading to epileptic encephalopathy, developmental delay and peripheral neuropathy. Respiratory failure has been described in some cases, caused by both central and peripheral neurological involvement. In published cases, the genetic diagnosis and initiation of appropriate therapy were rapidly performed following respiratory failure and led to a rapid recovery of respiratory insufficiency within days. Here, we present two infantile‐onset cases of isolated remethylation defects, cobalamine (Cbl)G and methylenetetrahydrofolate reductase (MTHFR) deficiencies, which were diagnosed after several months of respiratory failure. Disease modifying therapy based on hydroxocobalamin and betaine was initiated and shows a progressive improvement and enabled weaning off respiratory support after 21 and 17 months in CblG and MTHFR patients respectively. We show that prolonged respiratory failure responds to conventional therapy in isolated remethylation defects, but can require a sustained period of time before observing a full response to therapy. Respiratory failure is a well‐described symptom in isolated remethylation defects. Respiratory failure in isolated remethylation defects responds well to conventional therapy, even when the therapy is started with prolonged delay after the start of the respiratory symptoms.1MTRR and the MTR genes respectively, synthesising the methionine synthase enzyme and the methionine synthase reductase, an accessory protein required to maintain methionine synthase bound to cobalamin in its active form.Isolated remethylation defects (IRDs) are rare inherited disorders caused by defective remethylation of homocysteine in methionine, which prevents the synthesis of S‐adenosylmethionine (SAM), the main substrate for the numerous and critical methylation reactions required in physiology.Respiratory insufficiency is a well‐recognised complication of these disorders, caused by involvement of both central and peripheral nervous systems.2We reviewed the long‐term evolution of two patients with IRD, who were genetically diagnosed after a prolonged period of respiratory failure with clinical details summarised in Table 33.1N 10–60), ammonia, creatine kinase, very long chain fatty acids, lactate, electroencephalogram). An elevated homocysteine level was reported at 107 μmol/L (N < 15) but not investigated further. Brain MRI was reported to be normal. At 11 months of age, the child was admitted to her local general hospital to start nutritional support via nasogastric tube. During this admission, she desaturated to 70% oxygen resulting in intubation and transfer to a tertiary paediatric intensive care unit.This patient is the second child of unrelated parents. She was born at term after an uneventful pregnancy and presented with failure to thrive, developing anorexia for milk and solid food from the age of 5 months. At 6 months of age, she was treated as an outpatient for a lower respiratory tract infection with amoxicillin‐clavulanic acid. Subsequently over some weeks she started to show increasing respiratory distress, with recession and head bobbing. In parallel, and although her neurodevelopment had been normal until 6 months of age, she showed evidence of neuro‐regression with progressive generalised hypotonia, reduced mobility, hypomimic face, reduced smiling, difficulty to grab objects and move her limbs. She became much sleepier. Initial investigations at 10 months of age were reported as normal (plasma amino acids including methionine at 19 μmol/L (3.2MTR gene c.3518C > T (p.Pro1173Leu)/c.3600del (p.Ile1201Leufs). By that time, the child had profound hypotonia with no deep tendon reflexes, muscular strength scored 1/5 in proximal limbs, 2–3/5 in distality, following with her eyes but not being able to move her head independently. She was entirely fed by NG tube and required ventilation support via nasotracheal tube.Chest X‐ray showed bilaterally flattened diaphragms with both diaphragms at equal levels Figure . A diaphN 10–60). Homocysteine levels fell from 107 and 137 μmol/L to 20 μmol/L (N < 15). After 12 months of therapy, she was started on subcutaneous injection of hydroxocobalamin increased progressively from 1 to 4 mg daily, and spaced out from 7 to 4 injections per week enabling stable methionine and homocysteine levels.Following diagnosis, she was started on hydroxocobalamin 1 mg/day intramuscularly, betaine citrate 250 mg/kg/day and calcium folinate 15 mg/day orally. Methionine supplementation was started at 200 mg/day orally for 3 weeks then increased to 300 mg/day. Methionine levels normalised rapidly from 9 μM pretreatment to 40–68 μmol/L  and positive end expiratory pressure (PEEP) of +12 and +6 cmH2O respectively. At 3 months post‐initiation of therapy Figure , further3.4After 3 weeks of therapy, the child was moving all four limbs with increased muscular strength to score 3–4/5. She was starting to roll over, became more interactive with her environment, wanting to grab objects, smiling frequently with various facial expressions. After 20 weeks of therapy, she was able to sit, interacting very well, waving, clapping, speaking single words and tolerating some oral feeds. At 2 years of age, she was walking independently and speaking 10 words. At 4 years of age, she has caught up in regards of her motor development and communication. She is performing the same outdoor activities as any child of her age like swimming, cycling or jumping in a trampoline. She showed a mild speech delay in the 12 months following her decannulation, which could be attributed to her prolonged tracheostomy. This has now resolved fully resolved and she is speaking complex sentences with appropriate vocabulary for her age.Growth: At birth, her weight measured on the 96th centile, length on the 86th centile and head circumference on 80th centile. At initiation of therapy, her weight, height and head circumference were on the 3rd, 75th and 10th centiles respectively. At most recent follow up aged 4 years, her recent weight, height and head circumference were on 27th, 35th and 5th centiles respectively.44.1This patient is the first child of non‐consanguineous parents, who originate from the same village in Turkey. Her mother suffers from Familial Mediterranean Fever and had been treated for Hodgkin's lymphoma. The patient was born at 41 + 5 weeks gestation by spontaneous vaginal delivery in the UK following an uneventful pregnancy. She weighed 3.33 kg at birth. She was admitted for 5 days to the neonatal unit for neonatal respiratory distress and suspicion of infection, requiring oxygen supplementation and antibiotics, which rapidly resolved.At 1 month of age, the infant travelled to visit family in Turkey. Concomitantly she developed epileptic spasms, which precipitated hospital admission. An electroencephalogram performed revealed an epileptic encephalopathy and she was commenced on levetiracetam, vigabatrin, phenobarbitone and pyridoxine. She progressively developed poor feeding, signs of neuro‐regression, became lethargic and hypotonic with reduced interaction and eye contact. A video fluoroscopy performed at 2 months of age was complicated by aspiration pneumonia. She was intubated and admitted to the paediatric intensive care unit Figure . Extubat4.2MTHFR gene. Plasma biomarkers showed elevated homocysteine at 297 μmol/L and low methionine at 7 μmol/L. She was started on betaine 450 mg/kg/day and calcium folinate 15 mg daily. This enabled a decrease of total homocysteine to 70–80 μmol/L and normalised plasma methionine levels. However CSF 5‐methyltetrohydrofolate levels remain undetectable  at 4 years of age.At 20 months of age, whole genome sequencing identified MTHFR deficiency with homozygous pathogenic intronic mutation c.1753‐18G > A in the 4.3Patient was intubated and ventilated from 2 months of age. A tracheostomy was placed at 7 months of age. The patient sustained a total of 22 months of invasive ventilation. Four months after initiation of betaine and calcium folinate therapy, her respiratory function improved and she was weaned from ventilatory support at 24 months of age. At 4 years of age, stimulation of the phrenic nerve revealed reproducible responses suggesting at least partial innervation of the diaphragm bilaterally.4.4Following seizures at 1 month of age well‐controlled by anti‐seizure medication, the patient remained seizure‐free until the age of 2 years. Focal complex seizures were well controlled by rescue benzodiazepine as well as regular levetiracetam, and she was weaned off phenobarbitone. She showed severe global developmental delay with slow developmental progress. She acquired unsupported sitting at the age of 3.5 years. At the age of 3 years and 8 months, she could roll over but could not crawl or stand. She suffered from hyperkinetic movement of all four limbs. Her visual assessment showed large esotropia with roving eye movements, normal retinal function but flash visual evoked potentials were suggestive of rudimentary vision. Currently, she communicates by smiling to familiar faces, some babbling but has no speech and limited understanding.4.5At birth, her weight measured on the 42nd centile, length on the 95th centile and head circumference on 19th centile. At time of initiation of therapy, aged 20 months, her weight, height and head circumference measured 75th, 3rd and 85th centiles respectively. At most recent follow up aged 4 years her weight falls on the 9th centile, height on the 25th centile and head circumference on <0.4th centile.5Both cases present the typical infantile‐onset of two different IRDs, CblG and MTHFR deficiencies. In case 1 with CblG deficiency, an insidious onset of symptoms was observed with failure to thrive, food refusal followed by neuro‐regression and respiratory insufficiency. In case 2 with MTHFR deficiency, a subacute onset of neurological symptoms with epilepsy, encephalopathy and hydrocephalus was reported in infancy complicated by respiratory failure. Both cases showed prolonged respiratory failure requiring long‐term invasive ventilation via tracheostomy. Following genetic diagnosis, a disease modifying therapy based on hydroxycobalamin and betaine enabled a progressive response and the weaning of artificial ventilation.Respiratory failure is a well‐documented complication in infantile‐ and juvenile‐onset in both isolated and combined remethylation defects.The presented cases in our report showed full reversibility of respiratory failure despite prolonged periods of respiratory symptoms before initiation of therapy. Case 1 developed respiratory symptoms 5 months before initiation of therapy and was weaned off ventilatory support after 21 months of therapy and 22 months of artificial ventilation. Similarly, case 2 developed respiratory insufficiency 19 months before initiation of therapy and was weaned off ventilatory support after 4 months of therapy and 17 months of artificial ventilation. This differs from previous published cases of isolated or combined remethylation defects with respiratory failure where the recovery following initiation of therapy was shorter.These two reported cases also highlight the different neurological pathophysiology and response to therapy of the two disorders described. The patient with CblG deficiency presents with normal neurology at the age of 4 years whilst the patient with MTHFR deficiency suffers from severe developmental delay and is non‐verbal at a similar age. The natural history of untreated early‐onset MTHFR deficient patients results in profound neurocognitive impairment, severe cerebral atrophy and potentially hydrocephalus.In summary, we present two cases of infantile‐onset IRD with severe respiratory failure at diagnosis, requiring prolonged invasive ventilatory support, who demonstrated complete reversibility of respiratory symptoms on disease modifying therapy. This provides essential information for counselling patients and families at diagnosis regarding long‐term prognosis.Julien Baruteau is supported by funding from the United Kingdom Medical Research Council Clinician Scientist Fellowship MR/T008024/1 and NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.The authors have no conflict of interest to declare.Written consent for publication was obtained from patients' parents.


Whether estimated glomerular filtration rates (eGFRs) by differing biomarkers are differentially associated with mortality or whether the associations differ by diabetes status remains unclear, especially in Chinese population.2), modestly declined (60 to <90 mL/min/1.73 m2), and chronic kidney disease (CKD) (<60 mL/min/1.73 m2) groups. Cox proportional hazards models were used to estimate hazard ratio (HR) of all‐cause mortality associated with each eGFR.We included 6995 participants without diabetes (mean age: 60.4 years) and 1543 with diabetes (mean age: 61.8 years). Each eGFR measure was divided into normal  and eGFR creatinine (eGFRcr)‐cys  but not by eGFRcr were associated with higher risk of mortality. Among diabetes, all measures of modestly declined eGFR  and CKD  were associated with higher risk of mortality. Regardless of diabetes status, an addition of eGFRcys or eGFRcr‐cys to traditional risk factors lead to a larger improvement in the prediction of all‐cause mortality risk than adding eGFRcr.The association of eGFR with mortality risk appeared to be varied by its measures and by diabetes status among middle‐aged and older Chinese, which needs to be considered in clinical practice. HighlightsAll three estimated glomerular filtration rates (GFRs)  were more strongly associated with risk of all‐cause mortality among individuals with diabetes than among those without diabetes.Regardless of diabetes status, eGFRcys and eGFRcr‐cys were more strongly associated with risk of all‐cause mortality than eGFRcr.Regardless of diabetes status, an addition of eGFRcys or eGFRcr‐cys to traditional risk factors lead to a larger improvement in the prediction of all‐cause mortality risk than adding eGFRcr, which needs to be considered in clinical practices. Notably, serum creatinine is not only affected by health status but also by other host factors such as age, muscle mass, and diet.Among individuals with diabetes, CKD is one of the most common complications and has been widely associated with increased risk of premature death.To fill the knowledge gaps, we used data from the China Health and Retirement Longitudinal Study (CHARLS), a nationally representative cohort study of middle‐aged and older Chinese adults, to investigate the relationship between eGFR estimated by creatinine, cystatin C, or a combination of both and all‐cause mortality according to diabetes status.22.1The CHARLS is a population‐based prospective cohort study of individuals aged 45 years and upwards and is currently ongoing in China. Participants were recruited using a multistage probability sampling procedure from 150 counties across 30 provincial administrative units.This study used data from the four waves of the CHARLS. To be included in the current analysis, the study participants had to meet the following criteria: (a) had measurements of serum creatinine and cystatin C levels at W1; (b) had information to define diabetes status at W1; (c) were aged 45 years or older at W1; and (d) were successfully followed up in at least one of the next three waves. Finally, 8538 participants were included for further investigation Figure .2.2For each participant, venous blood (over 92% were fasting) was taken by trained personnel and delivered to a nearby laboratory at 4°C. Whole blood and centrifuged serum samples were frozen at −20°C, delivered to the Chinese Center for Disease Control and Prevention within 2 weeks in Beijing, and stored at −80°C until evaluated in the laboratory of Capital Medical University. The boronated affinity HPLC was used to test glycated hemoglobin (HbA1c). Serum cystatin C and creatinine were measured at baseline (W1) using a particle‐enhanced turbimetric assay with a coefficient of variation (CV) of 5.0% between assays and a rate‐blanked and compensated Jaffe creatinine technique with a CV of 2.1% between assays, respectively. Serum major lipids including high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, triglycerides, and plasma glucose were all determined using an enzymatic colorimetric assay. The CHARLS research samples underwent quality control (QC). All QC sample test findings were within 2 SDs of the mean QC control concentrations. Details of the laboratory analyses have been reported elsewhere.2.32), modestly declined (60 to <90 mL/min/1.73 m2), and CKD (<60 mL/min/1.73 m2) groups.We used three eGFRs to assess kidney function. The Chronic Kidney Disease Epidemiology Collaboration (CKD–EPI) formulas were used to measure eGFRs considering serum creatinine, cystatin C, or a combination of both, in addition to age and sex Table .4, 202.42). Diabetes was considered present if participants were diagnosed with diabetes by a physician or had fasting plasma glucose ≥126 mg/dL or HbA1c ≥6.5% at the W1 survey. Hypertension was defined as systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg, or self‐report of physician‐diagnosed hypertension or use of antihypertensive drugs. In formation on diagnoses of other medical conditions such as hyperlipidemia, heart problems  and stroke was also collected.Information on various sociodemographic and lifestyle factors, such as age, sex, living standard, education level, marital status, residence status , current smoking status, alcohol drinking , was obtained by interviewer‐administered questionnaires. Respondents were asked: ‘How is your standard of living?’ Five responses were provided: very high, relatively high, average, relatively poor, and poor. Living standard was further divided into high (very high or relatively high), average, and poor (relatively poor or poor) groups. BMI was calculated as measured weight (kg) divided by measured height  were available in W2, whereas only vital status was recorded in W3 and W4. For participants who were recorded as being decreased at W2 survey, follow‐up time was the interval between W1 interview and the date of death. For participants who were recorded as being deceased at W3, follow‐up time was the interval time between W1 and W2 in addition to median of the period between W2 and W3. A similar approach was used to derive follow‐up time for participants who were recorded as being deceased at W4 (W1–W3 period plus median of W3–W4). For the remaining individuals, follow‐up time was defined as the interval between W1 and the last interview wave with follow‐up information.2.6Baseline participant characteristics were reported by eGFRcr‐cys classifications , separately for participants without or with diabetes. Continuous variables were represented as mean ± SDs and categorical variables as numbers (percentages). Where appropriate, the analysis of variance or Kruskal–Wallis tests were used to assess continuous variables, and the chi‐square or Fisher's exact tests were used for categorical variables.Multivariate Cox proportional hazard models were used to examine the relationship between eGFRs and all‐cause mortality among individuals without or with diabetes, with the normal group as the reference. Two models with different degrees of adjustment for potential confounders were created. Model 1 was adjusted for age and sex. Model 2 was adjusted for age and sex and additionally adjusted for living standard, education level, marital status, residence status, smoking status, drinking status, BMI, hypertension, hyperlipidemia, and history of cardiovascular disease . To assess the potential nonlinearity for the examined relationships, each eGFR measure as a continuous variable was further modeled using restricted cubic splines with five knots at 5th, 27.5th, 50th, 72.5th, and 95th percentiles of the distribution of each eGFR.2. In addition, among participants without diabetes, we performed a further analysis to examine different eGFR measures in relation to all‐cause mortality by the status of prediabetes at baseline. Moreover, among participants who had normal kidney function defined by eGFRcr (≥90 mL/min/1.73m2), we examined the relationship between the other two eGFR measures (eGFRcys and eGFRcr‐cys) and risk of all‐cause mortality using the aforementioned full model.We performed a sensitivity analysis to examine different eGFRs in relation to risk of all‐cause mortality after excluding participants with eGFR of <15 or ≥120 mL/min/1.73mp value of <.05 was considered statistically significant.Finally, we evaluated improvements in the prediction of all‐cause mortality risk after adding each of the eGFRs to the multivariable model including traditional risk factors. Model discrimination and reclassification were evaluated using areas under receiver operating characteristic curves (AUCs), continuous net reclassification improvement indices, integrated discrimination improvement index, and change in C index.33.1There were 6995 participants without diabetes and 1543 with diabetes in the final analysis. Mean age was 60.4 years among those without diabetes (46.5% were aged 60 years or older), and 61.8 years among those with diabetes (53.1% were aged 60 years or older). Regardless of diabetes status, participants with lower eGFRcr‐cys were older; were more likely to be male, smokers, and regular drinkers; had higher systolic blood pressure; and had lower triglycerides, BMI, and educational levels. Lower eGFRcr‐cys was also associated with higher likelihood of cardiovascular disease and hypertension . Thus, the associations between different eGFR measures and risk of all‐cause mortality were reported according to diabetes status.Over a follow‐up of 7 years, 677 deaths occurred among the 6995 participants without diabetes at baseline, and 215 deaths occurred among the 1543 participants with diabetes. There were significant interactions between each of the eGFR measures and diabetes status on risk of all‐cause mortality (2) defined by eGFRcys or eGFRcr‐cys and higher risk of mortality, with fully adjusted HRs of 1.71 (95% CI: 1.28–2.28) and 1.55 (95% CI: 1.17–2.07), respectively. CKD defined by eGFRcr was not significantly associated with risk of all‐cause mortality . Regarding eGFR measures, modestly declined eGFR (60 to <90 mL/min/1.73m2) was not associated with risk of all‐cause mortality among participants without diabetes  for eGFRcr, 2.99 (95% CI: 1.83–4.88) for eGFRcys, and 3.92 (95% CI: 2.35–6.53) for eGFRcr‐cys defined CKD. Modestly declined eGFRcr , eGFRcys , and eGFRcr‐cys  were also associated with higher risk all‐cause mortality, though the risk estimate for eGFRcys was not statistically significant (Table p for nonlinearity <.001) or eGFRcys (p for nonlinearity = .023) and risk of all‐cause mortality, while the association for eGFRcr‐cys was more linear (p for nonlinearity = .51) .Among those without diabetes, evidence for a nonlinear (L‐shaped) relationship was observed between eGFRcr  including traditional factors increased the AUC from 0.7530 (95% CI: 0.7160–0.7910) to 0.7650 (95% CI: 0.7280–0.8020), and the AUC was slightly higher when eGFRcys  or eGFRcr‐cys  was added to the multivariable model  regardless of diabetes status. Second, for each of the three eGFR measures, the relationship with mortality risk appeared to be modified by diabetes status, with substantially stronger associations among participants with diabetes than among those without. Third, an addition of eGFRcys or eGFRcr‐cys into the multivariable model that included traditional risk factors improved predictive value for risk of mortality, especially so among participants with diabetes.2, with fully adjusted HRs of 2.43 (95% CI: 1.16–5.08) and 5.77 (95% CI: 2.77–12.03), respectively.2) was associated with increased risk of all‐cause mortality among individuals with diabetes.Although the CKD‐EPI formulaOur study extended the previously reported associations in diabetes to a Chinese population by diabetes status, showing that each of the eGFR measures was more strongly associated with mortality risk among individuals with diabetes than among those without diabetes. Nevertheless, our findings further showed that eGFR based on cystatin C or a combination of creatinine and cystatin C was more predictive of mortality risk than eGFRcr regardless of diabetes status. In addition, even among individuals without CKD according to eGFRcr, both eGFRcys and eGFRcr‐cys remained significantly and positively associated with risk of all‐cause mortality Tables . FurtherGiven that the estimating equation incorporating both creatinine and cystatin C is more positively related with directly measured GFR than each of other two eGFR measures,Potential explanations for the differences in the relationship between eGFR and mortality risk by different eGFR measures remain unclear. Previous studies have shown that serum creatinine is widely affected by age, muscle mass, diet, and physical activity, particularly in the high‐to‐normal range, and thus may be of limited abilities to identify early renal impairment.The present study is the first to evaluate the associations of eGFRs by different biomarkers and risk of all‐cause mortality according to diabetes status among Chinese. The current study has several strengths, which include a prospective design, a nationally representative sample, centralized measurements of blood samples in one single accredited laboratory, and comprehensive adjustment for the related covariates. We also included other comprehensive analyses in this study such as the metrics for multiple predictive models and the assessment of the potential nonlinearity for the examined associations. Notwithstanding, several potential limitations to this study should be considered. As an observational study, the potential influence of residual confounding on our results cannot be fully excluded. Due to the difficult procedures, adverse reactions, high cost, and the lack of urinary albumin, the GFR was estimated but not directly quantified.5In conclusion, our findings suggest that the relationship between eGFR and risk of all‐cause mortality appears to be different according to its measures and is modified by diabetes status. The eGFR measure based on cystatin C or both biomarkers (eGFRcys or eGFRcr‐cys) is more strongly associated with and more predictive of all‐cause mortality than the eGFR measure based on creatinine. Moreover, irrespective of eGFR measures, the relationship between eGFR and mortality risk is substantially stronger among participants with diabetes than among those without, which needs to be considered in clinical practices.This study was partially supported by Suzhou science and technology development plan (Livelihood Technology) project (No SS202009). Dr. Fu‐Rong Li is supported by Chinese Postdoctoral Science Foundation (2022M721463).The authors declare that they have no competing interests.Data S1. Supporting information.Click here for additional data file.


Opuntia ficus indica (L.) Mill.] are known to contribute to the antioxidant and antimicrobial activities of the prickly pear. The present study aimed to evaluate the antioxidants and in vitro antimicrobial potential in the hydroethanolic extracts of different parts  of prickly pear. Different polyphenolic compounds were analyzed by using high‐performance liquid chromatography. The results indicated that cladode possessed a higher quantity of phenolics compared with that observed in fruit and pulp. The most important phenolic compound in high quantity was gallic acid (66.19 μg/g) in cladode. The 100% aqueous extract of cladode exhibited the highest antioxidant (92%) and antimicrobial activities against Salmonella typhi (3.40 mg/ml), Helicobacter pylori (1.37 mg/ml), Escherichia coli (1.41 mg/ml), and Staphylococcus aureus (1.41 mg/ml). Principal component analysis (PCA) indicated that antioxidant activity and minimum inhibitory concentration (MIC) responses had a significant negative correlation with each other. Overall, the current results provided basic data for choosing prickly pear cladode with high antioxidant capacity for the development and consumption of antioxidant‐based alternative medicines and value addition of formulated foods.Phenolic compounds in prickly pear [ Prickly pear fruit and pulp contained high total phenolic, flavonoid, and carotenoid contents with strong antioxidant potential. The prickly pear exhibited antimicrobial potential against various microbial strains. TPC, TFC, and TC exhibited a positive correlation with antioxidant activity, whereas MIC exhibited a negative correlation with that DPPH activity. Belay et al. . The antimicrobial activity of cactus pear extracts was tested against different bacterial stains  that are resistant to different drugs. The constantly increasing demand for neutraceuticals is paralleled by a more pronounced request for natural ingredients and health‐promoting foods. Opuntia ficus indica has gradually attained economic importance in agriculture and the wider international community through the United Nations (UN) Food and Agriculture Organization (FAO). Opuntia has several health‐beneficial properties so there is a need to explore possible nutraceutical compounds. The aim of the present study was to determine the minimum inhibitory concentration (MIC) against different microbes, antioxidant capacity using DPPH assay as well as the phytochemical compositions and polyphenolic contents of Opuntia ficus indica cladode, pulp, and fruit.Cactus pear contains bioactive compounds, such as quercetin, isorhamnetin, and kaempferol. These bioactive compounds have significant antimicrobial activity against different microbes including  Nenaah, . Food‐boy et al.  revealed22.1Red pink‐colored prickly pear fruits of the cultivar (Meyer) with cladodes were brought from Talagang district, Chakwal, Pakistan. The samples were carried to the laboratory of the Institute of Food Science and Nutrition, University of Sargodha, Sargodha. Fruits and cladodes were washed separately to remove dirty materials, air‐dried, and stored in air‐tight glass containers at 4–6°C for use in different analyses.2.2Fresh cladodes extraction was carried out according to Benayad et al.  with few2.3Fruit and pulp extraction was performed following the protocol of Abou‐Elella and Ali  with few2.4Quantitative phytochemical screening was done using a spectrophotometer  and high‐performance liquid chromatography .2.4.1TPC was determined following the protocol described by AOAC method number 2017.13. For the assay, 0.25 ml of sample was taken in test tubes, mixed with 1.25 ml 10% Folin–Ciocalteu reagent, and diluted with distilled water 10 times. This solution was mixed with 1 ml of 7.5% sodium carbonate and incubated for 30 min in the dark. The absorbance was measured at 765 nm by spectrophotometer (Shimadzu Instruments). The TPC was expressed as gallic acid equivalents in milligrams (mg GAE mg)/100 g of FW.2.4.23 reagent for 30 min in darkness. The absorbance was recorded at 430 nm by the spectrophotometer. A calibration curve was prepared using quercetin as the standard. The results were expressed as mg equivalent of quercetin (mg QE)/100 g of FW  were determined by spectrophotometry through the reported method of Braniša et al. . Total c2.4.4A0 is the absorbance of the control and A1 is the absorbance of the extract.Free radical scavenging activity was determined by using the method of Shen et al.  with min2.52O:acetic acid, 94:6, pH = 2.27), solvent B (acetonitrile 100%) were used as the mobile phase in chromatographic separation in which 15% solvent B (0–15 min), 45% solvent B (15–30 min), and 100% solvent B (35–45 min) were used with 1 ml/min flow rate. Then a UV–visible detector was used which has a wavelength (λmax) of 280 nm to separate phenolic compounds. Phenolic compounds were determined by comparing UV–visible spectra and the retention time of the peaks with UV–visible spectra and the retention time of standards. The quantification was performed by external calibration.Flavonoids and phenolics were quantitatively analyzed by HPLC as per the reported method of AOAC, 2.6Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Salmonella typhi ATCC 19430, and Helicobacter pylori NCTC 11637, were brought from the Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan.MIC was determined by the broth dilution method. This assay was performed using a 96‐well microtiter plate. Bacterial strains for MIC, that is, 8 CFU/mL) and used as standard inoculums for the antibacterial studies. One hundred microliters of broth were poured into wells of a microtiter plate. Sample dilutions  were prepared. From 1 to 11, columns were labeled with these dilutions. The sample was poured into columns from 1 to 11. Bacterial inoculum was prepared to the size of 104–105 CFU/mL. 5 μL of bacteria was poured into wells in columns 1–12. Plates were incubated at 37°C for 12–18 h. Growth was checked by naked eye evaluation.For the assay, bacterial strains were cultured in Muller‐Hinton agar (MHA—pH 7.2) at 370°C. The stock culture slants were maintained at 4°C. The bacterial isolates were cultured in a Muller‐Hinton broth at 370°C for 4–6 h. The turbidity of the broth culture was adjusted to 0.5  and Pearson's correlation were conducted to evaluate the strength of the relationship between antioxidants and MIC of different parts of Opuntia. Data analysis was executed using statistical software Past 3 and Statistics 8.1. All measurements were recorded in triplicate (n = 3) and expressed as ±SD. The p < .01 was considered to be statistically significant.All the experimental data were first tested for normality with Kolmogorov's test. Tukey's test was applied to calculate the significant differences in different parts of the plant. A general linear model (GLM) was performed to compare the effects of different extracts and parts of 33.1p < .05) in the TPC of cactus pear's cladode, pulp, and fruit between parts and solvent fractions  TPC values, whereas the lowest TPC value was exhibited by ethanolic extracts. A decreasing trend was observed among the solvent fractions with progressive change in ethanol concentration (p < .01). Regarding parts of cactus pear, it was observed that the statistically highest TPC value was found in the cladode part, whereas the lowest value was recorded in the case of pulp (p < .01).The results showed significant variation  different among different parts of the prickly pear plant. The highest TFC was observed in cactus pear cladode, whereas the lowest value was observed in pulp (p < .01). With respect to solvent fractions, it was found that aqueous extracts yielded the highest TFC, whereas the lowest value was found in the case of ethanolic extracts (p < .01).The results of TFC regarding the influence of parts of 3.3Opuntia cladode contained significantly (p < .05) the highest carotenoids, whereas the pulp exhibited the lowest carotenoids  with a corresponding decrease in ethanol concentration (p < .01). Aqueous extracts exhibited the highest total carotenoid contents, whereas the lowest total carotenoid contents were found in ethanolic extracts.s Figure ; Table 13.4p < .01). Among the solvent fractions, aqueous extracts showed the highest (p < .05) activity, whereas the lowest activity was observed in the case of ethanolic extracts (p < .01). A decreasing trend in DPPH free radical scavenging activity was observed with corresponding increases in the ethanol concentration within the solvent fractions.The results of the DPPH scavenging activity are presented in Figure 3.5Staphylococcus aureus followed by Helicobacter pylori, Escherichia coli, and Salmonella typhi .MIC of extracts was higher against i Figure . The ext3.6Staphylococcus aureus, whereas the lowest MIC was observed in the case of Salmonella typhi (p < .01 for microbes). With respect to cactus pear parts, cladode exhibited higher MIC against different pathogens followed by pulp and whole fruit (p < .01) for cactus pear parts.The results with respect to the interaction between microbes and cactus pear parts on MIC of hydroethanolic extracts of cactus pear cladode, pulp, and whole fruit are presented in Figure 3.7p < .01 for cactus pear parts). Regarding solvent fractions, it was observed that aqueous extract exhibited maximum MIC against various pathogens, while ethanolic extract contained minimum MIC (p < .01 for solvent fractions). A declining trend in MIC against tested microbes was observed within the solvent fractions.The results regarding the interaction between cactus pear parts and solvent fractions on the MIC of cactus pear cladode, pulp, and whole fruit are depicted in Figure 3.8Opuntia cladode, pulp, and fruit extracts. In the case of Opuntia cladode, 100% aqueous extract exhibited apigenin, caffeic acid, vanillin, tannic acid, quercitin, gallic acid, rutin, and benzoic acid in the highest amounts of 8.06, 6.01, 7.91, 6.67, 15.84, 125.58, 57.07, and 61.84 μg/g, respectively, while quercitin (2.78 μg/g), gallic acid (11.6 μg/g), and rutin (4.61 μg/g) were lower in pulp as presented in Table The characterization and quantification of phenolic components of prickly pear cladode, pulp, and whole fruit was carried out using HPLC. The results showed that more phenolic compounds were observed in cactus pear cladode followed by pulp and whole fruit Table . Dealing3.9Salmonella typhi, Escherichia coli, Helicobacter pylori, and Staphylococcus aureus and antioxidants, that is, carotenoids, flavonoids, DPPH, and phenolic compounds along the PC1. Salmonella typhi, Escherichia coli, and Helicobacter pylori showed almost similar responses. The variation in PC2 was associated with Staphylococcus aureus response. The results indicated that most of the solvent fractions of fruit are located far from the flavonoids, carotenoids, phenolics, and DPPH, whereas solvent fractions of cladode and fruit are closely clustered to microbes. Aqu 100%, Aqu 80%; EoH 20%, Aqu 60%; EoH 40%, and Aqu 40%; EoH 60% of fruit showed a negative correlation with that of cladode along the PC1 axis, while on this axis, Aqu 20%; EoH 80%, EoH 100% of fruit, Aqu 40%; EoH 60%, Aqu 20%; EoH 80%, EoH 100% of pulp were strongly correlated with each other showed a positive correlation with that of cladode. Aqu 40% and EoH 60% of fruit showed a negative correlation with that of pulp compared with cladode. Aqu 20%; EoH 80% and EoH 100% of fruit, Aqu 20%; EoH 80%, Aqu 60%; EoH 40% and EoH 100% of pulp and Aqu 20%; EoH 80%, Aqu 40%; EoH 60% and Aqu 60%; EoH 40% of cladode were positively correlated with each other showed a positive correlation with that of pulp.Principle component analysis (PCA) was used to determine the correlation between antioxidant activity, MIC, and the various hydroethanolic extracts. PCA bi‐plot showing the relationship between antioxidant activity and microbes is presented in Figure 4Opuntia ficus indica contained about 54.33 ± 2.51 mg GAE/100 g, whereas Opuntia streptacantha exhibited TPC of about 104.66 ± 1.52 mg GAE/100 g DW. Teresita et al. . Larbi et al. (2017) mentioned that 70% ethanolic extract of cladode contained quercitin, gallic acid, tannic acid, apigenin, caffeic acid, and vanillin. The findings of the current research are in close association with the work of previous researchers  who concluded that Opuntia cladodes are a rich source of bioactive components which can play an important part in the human diet. Additionally, the variation observed in polyphenols among different fractions is affected by extraction time and temperature as reported by Robards  higher efficiency followed by ethanolic extracts against all tested pathogenic bacteria as indicated in Figure Opuntia is high in water. Mukonowenzou et al.  bacteria is famous due to its high sensitivity to phenolic extracts. Generally, Gram (−) is more resistant to bactericidal polyphenols than Gram (+) bacteria. According to Taguri et al. (Staphylococcus aureus (192 μg/ml) are more susceptible to polyphenols followed by Salmonella typhi (795 μg/ml) and Escherichia coli (1519 μg/ml). The present findings are in conformity with the results of Taguri et al.  pulp and fruit as a source of natural antioxidants. In general, HPLC data showed that prickly pear contained a high quantity of bioactive compounds. Opuntia cladode contained high amounts of total phenols, total flavonoids, and total carotenoids and also exhibited strong antioxidant potential. Regarding prickly pear whole fruit, the aqueous extract exhibited the highest amounts of rutin (7.43 μg/g), quercitin (3.41 μg/g), gallic acid (84.74 μg/g), syringic acid (23.54 μg/g), whereas ethanolic extract showed the low quantity of rutin (0.40 μg/g), quercitin (0.19 μg/g), gallic acid (29.22 μg/g), and syringic acid (5.18 μg/g). Moreover, prickly pear possesses effective antibacterial bioactive constituents against multi‐drug‐resistant bacteria and can be used for the prevention of different infectious diseases. The correlation analysis demonstrated phenolic contents, flavonoid contents, and carotenoids exhibited a substantial positive relationship with DPPH in an increasing manner. In this context, cladode is a valuable source of health‐promoting compounds, fulfilling concurrently the promising antioxidant activity that can be utilized virtually as food complements, to tardiness lipid oxidization and healing from particular ailments via its free radicals scavenging ability. It would be interesting to conduct more research to inspect the role of bioactive components which responsible for these activities. Hence, more studies are necessary to estimate the antioxidant and antimicrobial efficiencies of their individual purified fractions. Furthermore, it was revealed in the current study that the concentration of phenolic compounds and antioxidant activity in prickly pear extracts is sufficient to be considered a possible source of antioxidant supplements. In general, it may have the potential to contribute to better bioactive compounds contents in the diet of children, mothers, and adolescents to combat mineral deficiency problems and prevent many diseases including osteoporosis and cardiovascular disorders. The demand for fortified foods in the market is huge. Hence, prickly pear extracts fortification in foodstuffs is a convenient way to eradicate malnutrition. This abundant tree in Pakistan can become a great source of income for the nation if this potential for highly nutritional food is exploited by industries and researchers.The present work was focused on evaluating the resultant extracts obtained from the extraction of prickly pear (There was no funding received for this study from any funding organization.The authors declare that they have no conflict of interest to disclose.