Poria cocos (PC) and Alismatis rhizoma (AR) serve as a potential treatment. A systematic approach based on transcriptome sequencing analysis and bioinformatics methods was developed to explore the synergistic effects of PC–AR and identify major compounds and potential targets. The phenotypic characteristics results indicated that the high dose (4.54 g/kg) of PC–AR reduced total cholesterol (TC), elevated high-density lipoprotein cholesterol (HDL-C) levels, and improved hepatocyte morphology, as assessed via hematoxylin and eosin (H&E) staining. Transcriptomic profiling processing results combined with GO enrichment analysis to identify the overlapping genes were associated with inflammatory responses. The cytokine-cytokine receptor interaction pathway was found as a potential key pathway using geneset enrichment analysis. Core enrichment targets were selected according to the PC–AR's fold change versus the model. Real-time quantitative PCR analysis validated that PC–AR significantly downregulated the expression of Cxcl10, Ccl2, Ccl4, Cd40 and Il-1β mRNA (P < 0.05). Molecular docking analysis revealed the significant compounds of PC–AR and the potential binding patterns of the critical compounds and targets. This study provides further evidence that the therapeutic effects of PC–AR on hyperlipidemia in rats through the regulation of inflammation-related targets.Hyperlipidemia, a common metabolic disease, is a risk factor for cardiovascular diseases,  HLP contributes to the risk of arteriosclerosis, stroke, diabetes, and renal failure3, and has begun to affect younger generations, primarily because unhealthy lifestyles5. Therefore, the prevention and treatment of HLP warrants considerable attention.Hyperlipidemia (HLP), a lipid metabolism disorder, is an umbrella term for the abnormal elevation of total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) or a reduction in high-density lipoprotein cholesterol (HDL-C)7. Notably, statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA), are effective in eliminating LDL-C from the blood9. However, the long-term use of statins has various side effects, such as cataracts, diabetes, muscle problems, and drug tolerance11. Therefore, alternative treatments for HLP are urgently needed.Lipid regulators were once considered the gold standard for dyslipidemia12. Poria cocos (PC) is the dried sclerotia of the fungus Poria cocos (Schw.) Wolf, and Alismatis Rhizoma (AR) is the dried rhizome of Alisma plantago-aquatica subsp. orientale (Sam.) Sam13. These herbs were first recorded in Shennong’s Classic of Materia Medica and have been used for over 2000 years. According to Traditional Chinese Medicine (TCM) theory, the combination of PC and AR has the effect of invigorating the spleen and eliminating excessive moisture. PC and AR are commonly used collaboratively in prescriptions such as Wuling powder, Fuling Zexie decoction, and Liuwei Dihuang pills14. Wuling powder effectively reduced the weight and degree of obesity in patients with HLP15. Fuling Zexie decoction and Liuwei Dihuang pills improve serum lipid levels and protect the vascular endothelium18. In a complex network analysis of 453 medical cases, PC and AR were found to be key TCM pairs used to treat wet syndrome19. Studies on the active components of PC and AR have provided a basis for their combined use. Pachymic acid, pachymaran and other compounds extracted from PC have lipid-lowering, liver-protective, and anti-inflammatory effects22. Li et al. reported that 9 of 87 triterpenes with a high mean impact value had a significant lipid-lowering effect23. The main terpenoid monomers extracted from AR, 24-acetazenol A, 23-acetazenol C, and epoxisenol have anti-lipidemic effects on NAPCD cells24. At present, the use of these two herbal medicines individually is uncommon. To further investigate the synergistic therapeutic effects and underlying mechanism of the herbal pair PC–AR, this study integrated animal experiments, liver transcriptome technology, network-based analysis and molecular docking to identify potential disease biomarkers and candidate targets for HLP therapy  rats were obtained from the Experimental Animal Science and Technology Development Co., Ltd., affiliated with Southern Medical University (license number: SCXK 2011-0015). The rats were housed in an SPF-grade barrier environment at an ambient temperature of 21–25℃ with 50–60% humidity and maintained on a 12-h light/12-h dark cycle in the Laboratory Animal Center of Guangzhou University of Chinese Medicine . All animals protocols followed the Regulations of Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine and were approved by the Animal Ethics Committee . The entire research process including experimental procedures and data analysis, met the Essential 10 and Recommended Set of ARRIVE Guidelines 2.0 criteriaThe following equipment was used: a multifunctional microplate reader , paraffin embedding machine , automatic dehydrator , paraffin slicing machine ; optical microscope , vertical electrophoresis system , and automatic chemiluminescence imaging system .26.After one week of acclimatization, 40 rats were fed a high-fat diet (HFD) for four weeks. The HFD consisted of 52.2% maintenance feed, 0.4% premixed feed, 20% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 10% casein, 0.6% dicalcium phosphate, and 0.4% mountain flour. The rats in control group (n = 10) were fed a chow diet. At the end of the fourth week, after the rats had fasted for 12 h, blood lipids indices, including TC, TG, LDL-C, and HDL-C, were analyzed to evaluate whether the model was successfully established. The model evaluation criteria were assessed as described previouslyChinese Pharmacopoeia (2015) section I–IV. A 15-g PC and 10-g AR mixture was pre-soaked in distilled water for 20 min and then extracted twice with ddH2O , producing a 0.454 g/mL crude drug extract. The extract was then diluted to 0.114 g/mL and 0.227 g/mL. All herbal solutions were stored at 4℃ until use.PC (batch number: 190301) and AR (batch number: 190701), were purchased from Zisun Chinese Pharmaceutical Co. Ltd. . The Inspection Certificate Reports of PC and AR  indicated that herbs’ traits, identification, check items, extracts, and content met the standards of 27 , is as follows:The computational formula for determining the rat equivalent dose, according to body surface area based dosingThe daily dose of PC–AR (25 g) for a person (70 kg) is 0.36 g/kg, which is equivalent to the 2.27 g/kg concentration used as the median dose for the rats (0.2 kg). The low and high doses of PC–AR were 1.14 g/kg and 4.54 g/kg respectively. The volume of the intragastric liquid administered daily to the rats was 1mL/100g.Before the intervention was implemented, 50 male SD rats were divided into five groups (10 rats per group): control, model, 1.14g/kg PC–AR, 2.27g/kg PC–AR and 4.54g/kg PC–AR groups. In the control and model groups, PC–AR was replaced with an equal volume of 0.9% sterile saline. After four weeks of treatment, all rats were sacrificed, and blood and livers were harvested for further analysis.28 and Lee’s index28 were calculated after the experiment the following formulae:The rats from all five groups were fasted overnight at the end of the PC–AR treatment period. The body weight (BW) of all rats was measured before anesthetization. The animals were anesthetized by an intraperitoneal injection (50 mg/kg) of 2% pentobarbital sodium . Body length measurement and blood samples collection were performed under general anesthesia. Thereafter, all the animals were euthanized for liver sample collection. After standing for 1–2 h, the collected blood was centrifuged at 4 ℃ and 3,500 rpm for 10 min. The upper serum was collected and stored in a refrigerator at 20 ℃. The excised livers were washed with cold normal saline, frozen in liquid nitrogen, and stored at −80˚C until use. The liver coefficientTC, TG, LDL-C, and HDL-C concentrations were determined at the end of the fourth and eighth weeks using blood lipid detection kits provided by the Nanjing Jiancheng Biotechnology Company. Blood lipid detection was performed according to the manufacturer’s instructions.Collected rat liver tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated in gradient grade ethanol, cleared with xylene, embedded in paraffin, and cut into 4μm sections. The tissue slides were then dehydrated with xylene and gradient grade ethanol, incubated with hematoxylin for 5 min, stained with an eosin solution for 1–3 min, dehydrated again with gradient grade ethanol and xylene, and sealed with neutral gum. The liver cell nuclei (stained blue) and cytoplasm (stained red) were observed using a microscope.29 software. The mapping rates of the different samples against the reference database were between 73.76% and 83.81%. Fragments per kilobase per million fragments mapped values were calculated and used to normalize gene expression levels.Total RNA was extracted from the livers using TRIzol solution (Thermo Fisher Scientific) following the manufacturer’s protocol. RNA purity was assessed by determining the absorbance at 260 and 280 nm using a spectrophotometer . The RNA concentration was measured using the Qubit RNA Assay Kit along with the Qubit 2.0 Fluorometer , and RNA integrity was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies). A cDNA library was constructed using PCR amplification. The quality of the cDNA library was confirmed by quantitation, using a Qubit 2.0. The insert size was detected using Agilent 2100 istrument, and the library concentration was confirmed by quantitative PCR. All the samples were sequenced on an Illumina HiSeq 2500 platform. Raw data with a Q30 base percentage of over 86.09% were obtained. The obtained clean reads were blasted and mapped against the reference genome database  using the TopHat230 was used for data processing. All gene expression values were transformed into fold change 31. DEGs were defined according to the criteria |log2(FC)|> 0.585 and false discovery rate (FDR) 0.05. A volcano plot, processed using the ggplot2 package (v 3.4.1)32 was used to display the DEGs in both comparison sets based on the relationship between log10(FDR) and log2(FC). In addition, hierarchical clustering analysis was performed using the pheatmap package (v 1.0.12)33 to compare the DEGs among the groups. Raw sequencing data and processed data were uploaded to the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE212771.The R studio software (v 1.1.463)http://www.geneontology.org/), falling under three categories, molecular function (MF), cellular component (CC), and biological process (BP). Gene numbers were calculated for every term, and significantly enriched GO terms were defined using a hypergeometric test. The calculated P-value were subjected to false discovery rate (FDR) correction, with Q ≤ 0.05 used, as a threshold. GO terms that met this criterion were defined as significantly enriched.Gene ontology (GO) enrichment analysis was performed to determine the main mechanisms involved in the occurrence and treatment of HLP. First, all DEGs were mapped to GO terms in the GO database (rattus norvergicus was acquired from the Kyoto Encyclopedia of Genes and Genomes (KEGG)34 database and processed as a GMT file using R software (v 4.0.2)30. Other files containing the gene expression data in .txt format and the phenotype labels in .cls format for the model vs. control and the model vs. PC–AR groups were loaded into the GSEA (v 4.3.2)35. Finally, following standard protocols, 1,000 permutations were performed for each analysis to identify enriched pathways based on gene expression.GSEA was performed to verify which biologically significant gene sets were significantly associated with the different groups of samples. The annotated gene set for −△△CT. Primer sequences used in this study are listed in Supplementary Material 2.The RT-qPCR was used to detect the expressional levels of candidate targets. Total RNA was extracted from rat liver tissues using a lysis buffer with a Tissue RNA Purification Kit  according to the manufacturer’s instructions. After determining the concentration and purity of the RNA using a nucleic acid quantitative analyzer , reverse transcription was conduceted using a Color Reverse Transcription Kit . RT-qPCR was then performed with (ROX2 Plus) 2xColor SYBR Green qPCR Master Mix  on an fluorescence quantitation PCR system . Relative mRNA expression levels were normalized to β-actin (ACTB). The measurement results were expressed as 2http://www.tcmsp-e.com) 36according to oral bioavailability (OB) ≥ 30% and drug-like properties (DL) ≥ 0.18 criteria. Three-dimensional (3D) structure diagrams of the compounds were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). If no 3D structural diagram was available, two-dimensional (2D) structural diagram were used. The 2D structure diagram was converted to 3D using Chem3D software37. All structure diagrams were saved in PDB format. Next, the PDB structure files were imported into AutoDock4 (v 4.2.6)38 to add charging information and display rotatable keys. The files were saved in the required pdbqt format. The protein crystal structures corresponding to the core target genes were downloaded from the PDB database and imported into PyMOL39  to remove water molecules and impurities, following the addition of hydrogen atoms in the AutoDock4 and pdbqt formats. These compounds were used as ligands and proteins as receptors for molecular docking analysis. AutoDock4 was used to estimate the binding capacities of the molecules and targets. The results were visualized using PyMOL software (v 2.4.0 open source).Compounds contained in PC and AR were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . All data were analyzed using one-way analysis of variance (ANOVA) after the normality test. If the data met the condition of homogeneity of variance condition, the least significant difference (LSD) and Tukey’s post-hoc tests were used for three and four comparison groups, respectively. Welch’s analysis of variance combined with the Games–Howell post hoc test was applied under conditions of heterogeneity of variance when there was a significant difference between the groups. Statistical data are presented as the mean ± standard deviation. A 26. Permissions was obtained from the previous publisher. The medicine used in this study, Poria cocos and Alismatis rhizoma, differs from that used in the published literature. Additionally, the efficacy results, transcriptome data, comprehensive targets screening process and RT-qPCR validation results of PC–AR have not been published before, thus supporting for the originality of the study.Following the animal ethics principle of minimizing harm to animals, the control and model groups of rats used in this study were shared with the two groups previously described in published literatureA rat model of HLP was successfully established after four weeks. Different doses  of PC–AR were orally administered to the rats once per day for four weeks to determine the therapeutic effect of PC–AR on HFD-induced HLP. BW,the liver coefficient, Lee’s index, serum lipid levels and liver pathological morphology were compared after PC–AR treatment.P < 0.05) and an increase in the content of HDL-C (P < 0.05)  Fig. d-g. OnlyH&E staining Fig.  was usedFive control, five model, and six PC–AR samples, along with 11,436 genes, were included in this study. DEGs between different groups were identified, based on the criteria |log2(FC)|> 0.585 and FDR < 0.05 to demonstrate the molecular mechanisms of the effects of HLP and PC–AR, A total of 2502 HLP-related DEGs (1704 up-regulated and 798 down-regulated) were identified between the model and control groups . The top ten enriched BP terms . The core symbols characterized by a “yes” that enriched in the cytokine-cytokine receptor interaction pathway are listed in Supplementary Material 6. In addition, the top five targets, Cxcl10, Ccl2, Cxcl9, Ccl4 and Cd40, significantly down-regulated by PC–AR according to their FC values  and partly exerted the inhibition effect on the abnormal secretion of Cxcl9. Besides, PC–AR reversed the expression of Cd40 and Il-1β mRNA (P < 0.05), and slightly upregulated the Il-17rb mRNA, further indicating the inhibitory effect on liver inflammation.Combined RNA-sequencing and GESA analyses identified the cytokine-cytokine receptor interaction pathway as the key pathway in the hyperlipidemia treatment of PC–AR. HFD causes the chronic injury and secretion of inflammatory cytokines with over-expression of Cxcl10, Cxcl9,Ccl2, Ccl4, and Cd40 Fig. . C-C andMolecular docking analysis was applied to further explore the major compounds of PC–AR that play a role in its therapeutic effect on hyperlipidemia in rats and the potential binding patterns of the major compounds and key targets. A total of 13 compounds meeting the criteria of OB ≥ 30% and DL ≥ 0.18 were identified as major active compounds  , binding to the Cxcl10, Ccl2, Ccl4, Cd40 and Il-1β key targets .A schematic of the 3D representation of the molecular docking model with the lowest binding energy for each target to which the compound binds is shown in Fig. 42. This HLP model was the most consistent with the clinical characteristics of human HLP43. At the end of the modeling period, compared with those in the control group, the levels of TC and TG in the model group increased, and the HDL level decreased (P < 0.01), based on the criteria for establishing a mixed HLP animal model published in the “Evaluation Method of Auxiliary Hypolipidemic Function”44. After four weeks of high-dose PC–AR administration, serum TC level was reduced, and HDL-C level was significantly elevated (P < 0.05). Lee’s index and liver morphology also indicated the therapeutic efficacy of PC–AR. However, the dose–effect relationship was not clearly defined, and further research using more samples is required. To explore the targets and mechanism of action of PC–AR, transcriptomic analysis was performed. PC–AR improved the phenotypic characteristics of rats with HLP and altered gene expression. A total of 622 DEGs were identified as effectively regulated genes; these genes exhibited opposite expression patterns in the model group, compared to those in the other two groups.The HFD-induced HLP model used in this study was established using our previously developed modeling method), chemokine receptors (Ccr7 and Ccr5), cytokine receptors  and tumor necrosis factor ligand superfamily (Tnfsf10 and Tnfsf13). It is best known for the ability of chemokines to induce cell migration and is generally thought to be involved in all protective or destructive immune and inflammatory responses45. The enrichment results indicate a close relationship between dyslipidemia and inflammatory disorders, providing a clue for PC–AR achieving a inflammatory response regulation simultaneously with dyslipidemia intervention.Gene annotation and functional analysis of the 622 overlapping genes were performed to better understand the mechanism underlying the effect of PC–AR on HLP. GSEA of the 622 DEGs strongly indicated that PC–AR treatment of HLP in rats may function, in part, by regulating the cytokine-cytokine receptor interaction pathway, which is closely related to the inflammatory response. The core targets enriched in this pathway  included chemokines  that interact with toll-like receptors49. In addition, the synthesis of these cytokines is regulated by the ligation of CD40 on monocytes and macrophages which upregulates the production of matrix metalloproteinases (MMPs) and nitric oxide (NO)50. IL-17RB is the receptor of IL-25, a member of the IL-17 cytokine family, which constitutes the IL-25 signaling that develops beige adipose tissue. It was reported that the administration of IL-25 may increase beige fat and elevate adipose tissue thermogenesis in the way of alternately activating macrophages (from M1 to M2 polarization)51. In the present investigation, the expression of Cxcl10, Cxcl9, Ccl2, Ccl4, Il-1β and Cd40 mRNA increased and Il-17rb mRNA decreased in HFD-fed rats, suggesting that long-term HFD feeding induces an inflammatory response in the liver that gives rise to the production of pro-inflammatory cytokines. Our findings revealed that PC–AR administration reduced the expression of pro-inflammatory cytokines. This indicates the herbal pair PC–AR exerted the immunemodulatory effect on the liver response induced by high-fat diet exposure. At the same time, these results also suggest that PC–AR may affect the polarization of macrophages, which needs further investigation in the future.When an overabundance of free fatty acids impairs the storage capacity of adipose tissue, it leads to excess deposition of free fatty acids in the form of triglycerides in non-adipose tissue, resulting in lipotoxic damage to the tissue. Lipotoxic damage occurs in hepatocytes, priming immune cells, and inducing cytokines and chemokines release, ultimately leading to an immune cascadePoria cocos possess anti-inflammatory, immunomodulatory, anticancer and antihyperglycemic pharmacological properties52. In this study, the binding energy of trametenolic acid (one of Lanostane-type triterpenes) with Cxcl10 was −7.15 kcal/mol; the docking results with the other four targets are less than −5.0 kcal/mol, showing that this active compound has a good affinity with these targets. Alismatis rhizome, which contain more than 200 bioactive components (terpenoids in particular), have anti-inflammatory and antioxidant properties53. Aliso B 23-acetate and alisol C monoacetate, the active compounds extracted from Alismatis rhizome, were predicted to have a good affinity with Ccl2 and Cd40. Other components interacted well with these regulated targets . As shown in the current study, the treatment of HLP by PC–AR works through the effects of multiple components on multiple targets, the docking results provide predictive function for the subsequent selection of major core active ingredients. Previous studies have shown that the monomeric components extracted from isolated Poria cocos or Alismatis rhizome have the the function of regulating blood lipids55, and the current results after combination use suggest that PC–AR is associated with improvements in blood lipids, liver damage, and cytokine regulation, it is regrettable that a direct comparison between monotherapy or monomers and combined ones has not yet been made to prove the synergistic effects.Accumulating studies have elucidated that There are some limitations to our study. First, the active compounds of PC or AR are collected from databases and did not reflect all components of PC–AR, and future studies should apply advanced technologies to identify the certain components of PC and AR when mixed for the water extarction. Further, the water extraction methods should be compared with other methods to identify the extraction efficiency of the water extraction. Lastly, more comparison groups need to be set up in the future to compare the difference between Poria cocos or Alismatis rhizome alone and the combination of PC–AR.The improved serum lipid levels, Lee’s index, and liver pathological histology of rats in the PC–AR group indicate that PC–AR is a promising candidate for treating HLP. In addition, bioinformatics analysis of DEGs in rat livers suggested the potential of PC–AR to suppress HFD-induced immune and inflammatory responses. Cxcl10, Ccl2, Ccl4, Cd40 and Il-1β may be key targets of PC–AR in treating HLP.Supplementary Information 1.Supplementary Information 2.


P < 0.05) and provided adrenalectomy opportunities to 69% of patients judged through immunoassay to have bilateral disease. The secretion ratios  of aldosterone, 18-oxocortisol, and 18-hydroxycortisol were novel indicators for identifying unilateral PA. The 18-oxocortisol secretion ratio of ≥0.785‰ (sensitivity/specificity: 0.90/0.77) at pre-ACTH and aldosterone secretion ratio of ≤0.637‰ (sensitivity/specificity: 0.88/0.85) at post-ACTH enabled optimal accuracy for predicting ipsilateral and contralateral disease, respectively, in robust unilateral PA. LC–MS/MS improved the success rate of AVS and identified more unilateral diseases than immunoassay. The secretion ratios of steroids can be used to discriminate the broad PA spectrum.Adrenal venous sampling (AVS) is the gold standard for identifying curable unilateral aldosterone excess in primary aldosteronism (PA). Studies have demonstrated the value of steroid profiling through liquid chromatography–tandem mass spectrometry (LC–MS/MS) in AVS interpretation. First, the performance of LC–MS/MS and immunoassay in assessing selectivity and lateralization was compared. Second, the utility of the proportion of individual steroids in adrenal veins in subtyping PA was analyzed. We enrolled 75 consecutive patients with PA who underwent AVS between 2020 and 2021. Fifteen adrenal steroids were analyzed in peripheral and adrenal veins through LC–MS/MS before and after adrenocorticotropic hormone (ACTH) stimulation. Through selectivity index that was based on cortisol and alternative steroids, LC–MS/MS rescued 45% and 66% of failed cases judged by immunoassay in unstimulated and stimulated AVS, respectively. LC–MS/MS identified more unilateral diseases than did immunoassay (76% vs. 45%,  Primary aldosteronism (PA) is the most common cause of secondary hypertension, and it is surgically curable if unilateral excessive aldosterone secretion is identified. Adrenal venous sampling (AVS) is the gold standard for distinguishing unilateral aldosterone sources from bilateral ones in PA. However, only a minority of patients with PA who undergo AVS are eventually referred for unilateral adrenalectomy . In addiNevertheless, research on the overall performance of LC–MS/MS-derived SI and LI relative to immunoassay remains limited. Herein, we compared LC–MS/MS and immunoassay in terms of their AVS success rates and lateralization results. In addition, LC–MS/MS can be used to measure the ratio of individual steroid concentration to total steroid concentration in a single adrenal vein (AV). Therefore, our second goal was to establish indicators for predicting lateralization accurately by using unilateral AV samples.We retrospectively enrolled 80 patients with clinically suspected PA who underwent AVS between May 2020 and May 2021 and were followed up until February 2022 at National Taiwan University Hospital. We used prospectively collected data from the Taiwan Primary Aldosteronism Investigators (TAIPAI) database to standardize data collection . Five paPA screening, confirmation, and subtype differentiation were performed in accordance with the aforementioned TAIPAI protocol . In brieBecause Asian patients with PA have a high prevalence of autonomous cortisol secretion (ACS) , a low-dAVS was performed by a single radiologist with more than 10 years of experience in interventional vascular procedures. Blood samples were obtained simultaneously from bilateral AVs and the inferior vena cava (IVC). After unstimulated AVS was conducted, ACTH‐stimulated AVS was performed (sampling was performed for 15 min after an intravenous bolus injection of 0.25 mg cosyntropin). Catheterization was regarded to be successful when the SI (defined by AV/IVC cortisol concentrations) was ≥2 before ACTH stimulation and ≥5 after ACTH stimulation. The lateralization of PA was assessed through the LI . Unilateral PA was diagnosed if the LI was ≥2 before ACTH stimulation and ≥4 after ACTH stimulation. Contralateral suppression was indicated by a contralateral suppression index  of <1.Serum cortisol was measured using a chemiluminescent microparticle immunoassay . For cortisol, the intraassay and interassay coefficients of variation (CVs) were 3.3% and 3.4%, respectively, whereas the limit of detection (LOD) was 8 ng/mL. Plasma aldosterone was measured using radioimmunoassay kits in accordance with the manufacturer’s instructions . For aldosterone, the intraassay and interassay CVs were 7.7% and 8.4%, respectively, whereas the LOD was 0.007 ng/mL.We measured 15 types of adrenal steroids in a single assay through LC–MS/MS (an Agilent 1290 UHPLC coupled to a Sciex 6500 Qtrap); the measured steroids were as follows: aldosterone, cortisol, androstenedione, corticosterone, cortisone, 11-deoxycortisol, 21- deoxycortisol, 18-hydroxycortisol, 18-oxocortisol, dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS), 17-hydroxyprogesterone, progesterone, testosterone, and tetrahydrocorticosterone. Serum steroids were extracted and analyzed in accordance with the protocol used in a previous report [The Primary Aldosteronism Surgery Outcome consensus on clinical and biochemical outcomes was applied for patients with PA who underwent adrenalectomy .rs). Statistical analyses were performed using the one-sample t-test for continuous variables, Mann–Whitney U test, Kruskal–Wallis test (for nonparametric variables), and χ2 test . Paired variables were assessed using the Wilcoxon signed-rank test and McNemar’s test. A receiver operator characteristic (ROC) analysis was used to determine the optimal variable cutoff values for predicting PA lateralization. All analyses were conducted using the SPSS version 25 . All figures were created using the GraphPad Prism 9 software. For all statistical analyses, significance was indicated by a two-sided P value of <0.05.Unless otherwise specified, all data are expressed as means ± standard deviations or median and interquartile ranges. A Deming regression analysis and Bland–Altman plots were used to compare the results of immunoassay and LC–MS/MS. Normality and data variance were examined using the Shapiro–Wilk normality test. Relationships were assessed using the Spearman correlation coefficient  by immunoassay and LC–MS/MS and moderated positive relationships were observed between measurements of aldosterone  by immunoassay and LC–MS/MS. The Deming regression revealed a more favorable agreement for measurements of cortisol between the two methods. Overall, plasma cortisol and aldosterone were higher when LC–MS/MS was used than when immunoassay was used, and a significant difference (P < 0.001) between the peripheral and AVs was observed . In addition, progesterone-based SI was 2.2-fold higher in stimulated AVS  and stimulated AVS (88% vs. 85.3%). We further analyzed 13 types of alternative steroids and discovered that five types of steroids  had higher SIs than cortisol-based SIs when immunoassay was used; on average, they were between 1.8- and 2.3-fold greater and between 1.5- and 5.1- fold greater before and after ACTH stimulation, respectively  for both unstimulated and stimulated AVS. A pairwise within-patient comparison (for the 71 patients who underwent successful AVS) revealed that except for 18-oxocortisol, the LC–MS/MS-derived LIs normalized by cortisol and 12 alternative steroids were significantly higher  than the immunoassay-derived LIs. The difference was more pronounced (P < 0.001) in the unilateral PA that was classified on the basis of LC–MS/MS lateralization results . Furthermore, 57.1% of the patients with discrepant lateralization results (UB or BU) based on immunoassay were determined to have robust unilateral PA (UU) when LC–MS/MS was used (P < 0.0001). We observed a similar trend in patients with ACS. Overall, 54 patients (76%) and 32 patients (45%) were determined to have unilateral diseases  when LC–MS/MS and immunoassay, respectively, were used.Because of the LI differences between LC–MS/MS and immunoassay, a PA lateralization indicated discordance between assays Fig. . We clasIn total, 27 patients underwent unilateral adrenalectomy, and 20 cases of cortical adenoma and 7 cases of adrenal hyperplasia were confirmed through histological analysis. In addition, we assessed the postoperative outcomes of 11 patients who were followed up for at least 1 year. All of these patients achieved complete biochemical success, with 63.6% and 36.3% achieving complete and partial clinical success, respectively. For these 11 patients, two assays revealed concordant unilateral PA results for nine patients but discordant results for two patients. These two patients had unilateral PA according to LC–MS/MS and bilateral PA according to immunoassay, and both achieved the complete clinical and biochemical resolution of their PA.P < 0.0001). In patients with unilateral results only at pre-ACTH (UB), the secretion ratios of aldosterone and 18-oxocortisol were significantly higher in dominant AVs than in nondominant AVs . By contrast, in patients with robust bilateral disease (BB), the secretion ratios of all 15 steroids were not significantly different among bilateral AVs.The secretion ratio of steroids is the ratio of individual steroid concentration to total steroid concentration in the unilateral AV  . Throughentclass1pt{minimaWe compared the unilateral secretion ratios of aldosterone, 18-hydroxycortisol, and 18-oxocortisol in patients with unilateral ipsilateral disease, unilateral contralateral disease, and bilateral disease Fig. . The terThe ROC curves indicated the utility of unilateral aldosterone, 18-hydroxycortisol, and 18-oxocortisol secretion ratios for the diagnosis of robust unilateral PA (UU) Fig. . The optOur study revealed two major findings. First, we demonstrated that an LC–MS/MS assay has greater utility than the traditional immunoassay in enhancing the assessment of selectivity and discrimination of unilateral PA. Second, we revealed the diagnostic value of the unilateral secretion ratios of aldosterone and the hybrid steroids 18-oxocortisol and 18-hydroxycortisol for PA subtype classification. In addition, we examined the TAIPAI dataset of AVS studies and compared LC–MS/MS assay and immunoassay results for both unstimulated and ACTH-stimulated AVS in patients with PA.In line with the results of other studies , 8, 12, For lateralization ratios, the LC–MS/MS-derived LI of aldosterone that was corrected by 13 steroids was considerably higher than the immunoassay-derived LI of aldosterone. This finding is reflected in the first set of results obtained in our study, which revealed the differences in aldosterone measurements between immunoassay and LC–MS/MS, and these differences were particularly prominent in the low concentration range. The cross-reactivity of antibodies between aldosterone at low concentrations and other steroids at high concentrations can affect the accuracy of immunoassays in the measurement of aldosterone . ConsequIn contrast to the considerable decrease in the lateralization of the PA assessed by immunoassay, ACTH stimulation had a lesser effect on lateralization status when LC–MS/MS-derived LI was used. Studies have suggested that ACTH stimulation is a double-edged sword because it improves the success rate of cannulation but neutralizes the aldosterone step-up between bilateral adrenals by promoting contralateral aldosterone secretion , 14. HowThe second key finding of our study is that the secretion ratios of aldosterone, 18-hydroxycortisol, and 18-oxocortisol, as measured using LC–MS/MS, are novel indicators for unilateral PA. Traditionally, LI was the indicator used to assess the aldosterone excess; the assessment involved comparing the aldosterone concentrations of two adrenals. In the present study, we used the secretion ratio of aldosterone to assess aldosterone excess by comparing aldosterone concentrations to total steroid concentrations. These findings can be used to predict lateralization when only unilateral AVS is successful. For unilateral ipsilateral disease, all three steroids  exhibited diagnostic accuracy with AUCs of >0.8, whereas 18-oxocortisol and aldosterone exhibited the highest diagnostic power for unstimulated and stimulated AVS, respectively. For unilateral contralateral disease, only 18-oxocortisol and aldosterone exhibited significant suppression on the contralateral side. This suggested that a sufficient amount of 18-hydroxycortisol was derived from the contralateral zona fasciculata through CYP11B1 when CYP11B2 activity was suppressed . On the In several previous studies, attempts have been made to predict lateralization using one-sided AV samples –19. HoweThe secretion ratios of 18-hydroxycortisol and 18-oxocortisol may provide additional information for discriminating PA spectra. Several studies have reiterated the utility of these hybrid steroids in differentiating APA from BAH through the use of urine or serum samples , 20, 21.Few studies have explored the effect of low-grade cortisol concentration in AVS interpretation. A small single-center study, involving patients with both PA and ACS, reported that ACS had a limited effect on AVS interpretation after ACTH stimulation ; this fiIn addition to the aforementioned limitation regarding ACS subgroup analysis, the present retrospective study has several other limitations. First, this was a single-center study that examined an East Asian cohort; because of differences among ethnic groups, AVS protocols, and interpretation criteria, the present results should be generalized with caution. Second, our analysis of the PA spectrum was limited by the incomplete postoperative follow-up data and the pathologies that were based on hematoxylin and eosin histology instead of immunostaining. Further studies with complete outcome data and CYP11B1/ CYP11B2 immunostaining are required to clarify the PA spectrum link with steroid profiling upstream and the pathologic and clinical outcome downstream.LC–MS/MS assay optimizes the assessment of selectivity and lateralization in AVS, and it can identify more candidates for curable adrenalectomy than immunoassay can. In addition, the present study is the first to demonstrate the use of the secretion ratios of aldosterone, 18-oxocortisol, and 18-hydroxycortisol in unilateral AV as a discriminating marker for PA lateralization, which can be advantageous when only unilateral AVS is successful. Finally, the present study also verified that steroid profiling is promising for establishing a diagnostic method for the PA of varying levels of severity.Supplementary Methods


There is a lack of evidence guiding the surgical timing selection in pulmonary atresia with ventricular septal defect. This study aims to compare the long-term outcomes of different initial rehabilitative surgical ages in patients with pulmonary atresia with ventricular septal defect (PAVSD).From January 2011 to December 2020, a total of 101 PAVSD patients undergoing the initial rehabilitative surgery at our center were retrospectively reviewed. Receiver-operator characteristics curve analysis was used to identify the cutoff age of 6.4 months and therefore to classify the patients into two groups. Competing risk models were used to identify risk factors associated with complete repair. The probability of survival and complete repair were compared between the two groups using the Kaplan-Meier curve and cumulative incidence curve, respectively.p = 0.027) and right ventricle-to-pulmonary artery connection  were associated with increased probability of complete repair. The cumulative incidence curve showed that the estimated complete repair rates were 64% ± 8% after 3 years and 69% ± 8%% after 5 years in the younger group, significantly higher than 28% ± 6% after 3 years and 33% ± 6% after 5 years in the elder group (p < 0.001). There was no significant difference regarding the estimated survival rate between the two groups.The median duration of follow-up was 72.76 months. There were similar ΔMcGoon ratio and ΔNakata index between the two groups. Multivariate analysis showed that age ≤6.4 months (hazard ratio (HR) = 2.728; 95% confidence interval (CI):1.122–6.637; Compared with those undergoing the initial rehabilitative surgery at the age >6.4 months, PAVSD patients at the age ≤6.4 months had an equal pulmonary vasculature development, a similar probability of survival but an improved probability of complete repair. A total of 101 PAVSD patients undergoing the initial rehabilitative surgery, either through systemic-to-pulmonary artery shunt or right ventricle-to-pulmonary artery connection, between January 2011 to December 2020 at Guangdong Provincial People's Hospital were retrospectively reviewed and included in this study. PAVSD patients with concurrent complex intracardiac abnormalities or those who were not suitable for bi-ventricle repair were excluded from the study. Of 101 patients, those who were referred to our hospital early underwent the initial rehabilitative surgery in time after a full assessment of clinical conditions such as pulmonary artery development, MAPCAs distribution, and pulmonary flow. However, some were delayed in undergoing the initial rehabilitative surgery, predominantly due to the late referral to our hospital. As shown in The medical history, imaging examination, surgery information, and perioperative records were all extracted and collected. The McGoon ratio and Nakata index were calculated on CT imaging to assess the pulmonary vessel growth, as described by Jia et al. . The Nak2.2.The technique for establishing RV-PA connection was previously described in detail . BrieflyIn contrast, the establishment of a systemic-to-pulmonary shunt could be performed without the use of a cardiopulmonary bypass, including a central shunt and a modified Blalock-Taussig (B-T) shunt. The technique for the central shunt was described by Gates et al. in 1998 . BrieflyWhen satisfactory pulmonary artery growth was obtained, the complete repair would be performed by reconstruction of the right ventricle outflow tract using a valved conduit or patch as well as VSD closure.2.3.All patients were scheduled for regular follow-up visits at the outpatient clinic at 1, 3, 6, and 12 months after the rehabilitative surgery, followed by annual visits. CT would be performed if there was satisfactory pulmonary vessel growth or suspicious vessel stenosis. A recent phone contact has also been performed to investigate their latest clinical status including NYHA heart function class, complication, survival, complete repair, and so on. The date of the recent phone contact was November 29th, 2022. Those who never came to visit the outpatient clinic in the entire postoperative period or only came to visit the outpatient clinic for few times in the initial postoperative period and could not be contacted by the recent phone call were considered to be lost to follow-up. The endpoints were defined as death or complete repair during the follow-up. In-hospital mortality or follow-up mortality were defined as death before discharge and during the follow-up, respectively. The ΔMcGoon ratio/ΔNakata index was defined as the difference between the McGoon ratio/Nakata index before the complete repair and before the initial rehabilitative surgery.2.4.p < 0.001), which was therefore used to classify all the PAVSD patients into two groups (age ≤6.4 months vs. age >6.4 months). The student's t-test or Mann–Whitney U-test was applied to compare the continuous variables with normal distribution or abnormal distribution between the two groups. The chi-squared test was used to compare the categorical variables between the two groups. Competing risk regression analysis were performed for the complete repair (with death as a competing event). The postoperative survival was estimated by the Kaplan–Meier curve with the log-rank test. The complete repair was estimated by cumulative incidence curve with Gray's test. All statistical analysis was conducted using SPSS version 24.0. A p-value less than 0.05 was considered to be significant.Continuous variables were presented as means with standard deviations if normally distributed or medians with ranges if abnormally distributed. Categorical variables were presented as numbers with percentages. The receiver-operator characteristics (ROC) curve was created with the area under the curve (AUC) to determine the cut-off age. As shown in 3.3.1.p > 0.05). Compared with the elder group, the younger group showed significantly lower weight, BSA, presence of MAPCAs, McGoon ratio, and Nakata index (p < 0.05).The baseline clinical characteristics were summarized in the 3.2.p = 0.020) and longer length of hospital stay (p = 0.016). The mean follow-up time of the entire cohort was 72.76 months. The follow-up mortality was similar, with 4 and 6 deaths occurring in the younger group and the elder group, respectively. There was also no significant difference in the ΔMcGoon ratio and ΔNakata index between the two groups. Of note, inter-stage reintervention including percutaneous balloon pulmonary valvuloplasty, percutaneous pulmonary artery stent implantation, and shunt/conduit replacement were similar between the two groups . The overall 3-year and 5-year complete repair rates in the younger group were 64% ± 8% and 69% ± 8%, respectively, in contrast to 28% ± 6% and 33% ± 6% in the elder group, respectively. Additionally, Patients initially undergoing RV-PA connection also had a significantly higher probability of complete repair in contrast to those undergoing systemic-to-pulmonary artery shunt (p = 0.035). No significant differences were observed in terms of the probability of survival by age groups (In the e groups  or rehabe groups .3.4.p = 0.027), McGoon ratio , Nakata index , and RV-PA connection  were identified as independent factors associated with complete repair.The univariate regression analysis showed that age ≤6.4 months, weight, presence of MAPCAs, Nakata index, and RV-PA connection were predictors of complete repair . These p4.The ideal treatment and management for PAVSD patients, particularly regarding the determination of surgical timing, remains undefined. To the best of our knowledge, this is the first study to identify the cutoff age at initial surgery for PAVSD to evaluate its impact on clinical outcomes. In this study, our findings showed that PAVSD patients in the younger group had a significantly improved complete repair possibility than those in the elder group. What's more, despite a higher incidence of delayed sternal closure, the younger group had similar and comparable pulmonary artery growth and mortality compared to the elder group.In our study, the estimated complete repair rate in the younger group (median age: 1.90 months) was 64% ± 8% after 3 years and 69% ± 8% after 5 years, which was significantly higher than the 28% ± 6% after 3 years and 33% ± 6% after 5 years in the elder group (median age: 33.37 months). In general, our estimated complete repair rates were somewhat lower than those in the previous studies , 12. We There were no deaths in hospital. The follow-up mortality was also comparable between the younger group (10%) and the elder group (9.8%), which was consistent with the previous investigation –20. MacaFor Tetralogy of Fallot selecting for complete repair, it is recommended to perform surgical correction between the age of 3 months and 6 months with a Class IIa recommendation . Though p = 0.001). Which kind of rehabilitative approach for PAVSD is more ideal remains highly debated. Fan et al. compared the perioperative and postoperative outcomes between 44 patients with systemic-to-pulmonary artery shunts and 54 patients with RV-PA connections. They demonstrated that systemic-to-pulmonary artery shunt resulted in better early postoperative outcomes in terms of shorter intubation time, length of intensive care unit stay, and length of hospital stay (In the multivariate analysis for complete repair, RV-PA connection was a positive predictor (HR = 4.196; 95% CI = 1.782–9.883; tal stay . Zhao ettal stay , which wThere were several limitations in our study. First, the results should be cautiously interpreted due to the retrospective one-single-center design. Secondly, the initial pulmonary vasculature status was different between the two groups, which may overestimate the impact of early surgery on outcomes. In addition, a more comprehensive and targeted analysis for comparing reintervention between the two groups should be performed to address safety concerns in younger patients. Finally, our variable reflecting the cardiac function performance improvement was limited. New York Heart Association Classification, clinical status, and oxygen saturation will be investigated in the future.5.Despite similar pulmonary artery growth, PAVSD patients undergoing the initial rehabilitative surgery at the age ≤6.4 months had an improved possibility of complete repair without increasing risk of mortality and reintervention in contrast to those at the age >6.4 months. These findings aid in surgical timing selection, benefiting the evidence supplement of the management algorithm in treating PAVSD patients.


Dietary intake and body weight are important predictors of long-term health. However, few studies have focused on these topics in adults with genetic syndromes that have associated intellectual disability, such as Williams syndrome (WS).In adults with WS, describe predicted dietary intake, food-related problems, and associations between body mass index (BMI) and possible factors contributing to differences in weight status.n = 41).In this study of 82 participants , we cross sectionally investigated associations between BMI, predicted dietary intakes (Dietary Screener Questionnaire), food-related behaviors (Food-Related Problem Questionnaire), and anxiety . Longitudinal patterns of weight change were further studied in a subset (2 (range 16.7–55.5 kg/m2). Several components of dietary intake deviated from recommendations in the WS cohort. When compared with WS participants with either normal or overweight BMI, WS participants with obesity had reduced daily intake of fruits and vegetables of 0.15 cup equivalents (P = 0.049), while participants with underweight BMI had reduced daily intake of fruits and vegetables of 0.44 cup equivalents (P = 0.026) and additionally had reduced intake of dietary fiber of 2.12 grams per day (P = 0.019). A one-point increase in the ‘preoccupation with food’ sub-score was associated with a 0.57 unit increase in BMI (P = 0.16), while a one-point increase in the ‘takes and stores food’ sub-score was associated with a 0.72 unit increase in BMI. In the longitudinal weight subset, a weight gain group and a weight stable group were identified. The former was associated with increased ‘takes and stores food’ sub-score but not with dietary intakes.BMI variation was observed with median BMI of 27.3 kg/mWe observed considerable BMI variability. While few dietary intakes were associated with BMI, increased BMI and weight gain were associated with ‘preoccupation with food’ and with ‘takes and stores food’ behavior sub-scores. Variability in BMI and patterns of weight change over time were observed in a group of adults with Williams syndrome, a rare genetic disorder associated with intellectual disability.Dietary intakes of individuals with Williams syndrome deviated from national recommendations; however, few of the dietary intake components were related to BMI variability.Selected food-related problem behaviors were found to be associated with increased BMI or higher rate of weight gain.2 (classified as obesity) or a BMI < 18.5 kg/m2 (classified as being underweight). Less scientific attention has been paid to the issue of weight status among adults with intellectual and developmental disability (IDD), despite studies demonstrating an increased frequency and earlier onset of obesity compared with the general population  ≥30 kg/mpulation –7, whilepulation . The majpulation  and so fThe objectives of this study were to investigate BMI in adults with WS and to reveal possible factors contributing to differences in weight status classifications and weight gain in adults with WS.Our specific aims were to: 1) describe predicted intakes of selected dietary components in adults with WS and compare intakes with controls from National Health and Nutrition Examination Survey (NHANES); 2) analyze associations between BMI, predicted intake of dietary components, food-related behaviors, and anxiety in the WS study participants; and 3) for a subset of participants with WS, compare those with the highest rate of weight gain to those with stable weight over time to further explore possible factors associated with weight gain.www.williams-syndrome.org), The Williams Syndrome Patient & Clinical Research Registry (www.williams-syndrome.org/registry), Rally with Mass General Brigham , or by hard copy invitation sent to personal patients of one of the authors (BRP). Eligibility criteria included the following: males or females between the ages 18 and 70, diagnosis of WS, and availability of a parent or guardian to participate in the consent process and provide medical history.Individuals with WS and their families were recruited through a study announcement posted on websites of the Williams Syndrome Association  between October 2018 and February 2020 or at a Williams Syndrome Association Family Convention . Study recruitment was terminated at the start of the COVID-19 pandemic.2), normal (18.5–<25 kg/m2), overweight (25–<30 kg/m2) or obese (≥30 kg/m2)  . ParticiAll participants, with a family member or guardian, completed several surveys. For the purpose of this study, we developed a health and family history questionnaire, which covered topics such as past medical history, family history, weight status over time, and medication use at the time of the study.The Dietary Screener Questionnaire (DSQ), developed by the National Cancer Institute (NCI), calculates predicted mean daily intake (subsequently referred to as daily intake) using responses to 26 survey questions. Specifically, daily intakes are based on self-reported intake frequencies during the past month of multiple food and beverage item groups; frequency report options include: never, number of times last month, number of times last week, and number of times per day. Responses were analyzed using the current National Cancer Institute -recommended scoring procedures developed on data from the 2009–2010 NHANES cohort , 14. ThiN = 2,022 adults belonging to the 2009–2010 NHANES cohort were used as a control cohort  and U.S. Department of Health and Human Services (HHS) Dietary Guidelines for Americans (DGA) 2020–2025, Table 4-1 (page 96), for adults consuming a 1,800–2,400 calorie per day diet .Food and eating behaviors in the cohort were characterized using responses to the Food-Related Problem Questionnaire (FRPQ). This is a 16-item questionnaire scored on a 7-point Likert scale from 0 (never) to 6  indicating the frequency of the behavior, with a Likert score ≥1 indicating at least some frequency of the behavior. Results are summarized in subscores of ‘preoccupation with food’, ‘impairment of satiety’, and ‘composite negative behavior’. The ‘composite negative behavior’ can be further divided into subscores of ‘takes and stores food’, ‘inappropriate response’, and ‘eats inedible items’ –19. PicaThe Spence Children’s Anxiety Scale-Parent version (SCAS-P), a validated parent proxy-report survey , was useData from participants who authorized access to historic heights and weights from records in their primary care provider’s office were used to generate weight (kg) over time (months) trajectory plots. Individuals with two or more weight points at least 6 months apart were included in this analysis; the terminal weight point for each subject was measured during study participation.Variables were assessed for normality using the Shapiro–Wilk test; log transformation was applied to skewed data. DSQ daily intake for each dietary component group was calculated as described in Methods section.t-tests examined sex differences in daily intake in the WS cohort only.Independent samples t-test assessed for BMI and age differences between the WS and NHANES cohorts. Linear regression analyses controlling for age assessed for daily intake differences between cohorts; this was repeated in sex-separated models also controlling for age. Bonferroni correction for the eight multiple comparisons within the whole cohort as well as the sex-specific sub cohorts, was applied to adjust the significance level (P < 0.00625).Welch’s Logistic regression analyses adjusted for sex and age examined BMI category associations with DSQ daily intakes as follows: obesity versus normal weight and overweight, and underweight versus normal weight and overweight.FRPQ subscale scores were computed for each subject. Linear regression analyses adjusted for sex and age investigated associations between BMI and FRPQ subscale scores, and between BMI and frequencies of individual food behaviors.SCAS subscale/total scores were computed for each subject. Kruskal–Wallis tests were utilized to assess for differences between BMI categories  and mean SCAS subscale/total scores.A linear mixed-effects model utilizing the restricted maximum likelihood method with a diagonal covariance structure evaluated weight trajectories over time and the effects of covariates. The model included age, sex, and their interaction for fixed effects. Difference in subject starting weight was accounted for as a random effect of subject-specific intercept. An average weight change over time (kg/year) was obtained controlling for the fixed effects described here. Subject-specific slopes were obtained, and weight point specific residuals based on group mean revealed two distinct classes of weight trajectories for analysis: Group 1: ‘high gain trajectory’ was comprised individuals with a line of best fit slope of a magnitude in the top third of the cohort; and Group 2: ‘stable weight’ individuals, with a slope in the middle third of the cohort closest to zero and who did not experience two consecutive periods of gain or loss more than 2.5% of total body weight. Slopes from a third group of individuals were not used for analysis as they did not meet criteria for either Group 1 or Group 2 .t-tests investigated potential associations with birthweight.To explore possible factors associated with weight gain, chi-square analyses compared the two weight groups in factors such sex distribution, diagnosis of diabetes mellitus, self-reported current use of levothyroxine, or of weight-promoting medications as classified in Wharton et al.  and alsoData were analyzed using SPSS Statistics for Windows, version 24.0 . In analyses of food behavior data STATA version 16  was used. Figures were created using JMP Version 16 .in situ hybridization [FISH] or chromosomal microarray analysis [CMA]) in 79 of the 82 individuals while, in the remaining three, the diagnosis was confirmed clinically by one of the co-authors (BRP), an experienced medical geneticist.A total of 86 adults with WS consented to participate in this study. Thirty individuals took part in the tCRC at MGH, while 56 participated at a WSA Family Convention. Four participants were removed from all statistical analyses due to missing data for both dietary assessment and historic weight measurements; accordingly, the cohort available for detailed analyses consisted of 82 adults with WS. The diagnosis of WS was confirmed by genetic testing  (2), normal (18.5–<25 kg/m2), or obese (BMI ≥30 kg/m2) categories were comparable, there was a significantly higher percent of males with BMIs in the overweight range (BMI 25–<30 kg/m2: 37.1% M vs. 19.1% F)  . There w19.1% F) .2)  but were, on average, younger . Mean daily intakes for selected major food groups are shown in P < 0.001), and of calcium  were higher than in NHANES males. A similar non-significant trend was observed in females for dairy  and calcium . A combined sex analysis retained significance for both intake categories, though the effects were attributed primarily to the excess in WS males .Participants with WS as a group had a mean BMI comparable to NHANES controls (kg/mP < 0.001), sugar-sweetened beverage serving equivalents per day (P = 0.050), dairy (P < 0.001), dietary fiber (P = 0.001), and calcium (P < 0.001).Sex analyses within the WS cohort  demonstrComparison to national dietary recommendations from the 2020 Dietary Guidelines for Americans (DGA)  showed tP = 0.049). The group of WS participants with an underweight BMI (BMI < 18.5) when compared with the groups with normal and overweight, had a reduced mean intake of fruits and vegetables of 0.44 cup equivalents per day, ; they also had a reduced intake of dietary fiber of 2.12 g/day . No significant associations between BMI category and mean intake of dairy, added sugars or whole grains were detected.WS participants with obesity (BMI ≥ 30), when compared with WS participants who had either normal weight or overweight (BMI 18.5–<30), had a lower mean intake of fruits and vegetables of 0.15 cup equivalents per day,  of a maximum score of 18, while the total ‘impairment of satiety’ sub-score was 10.33 (SD 4.15) of a maximum score of 30. Examination of specific items that comprise the ‘impairment of satiety’ subscale reveals a high frequency of selected food behaviors. The majority of adults with WS (70%) were reported as displaying at least some frequency of the behavior, ‘if given the opportunity, eats more than a standard sized meal’. For the ‘takes and stores food’ subcategory, the total sub-score was 3.83 (SD 3.93) of a maximum score of 18; 87% of adults were reported displaying some frequency of helping themselves to food that they should not have, a specific item that comprises this subscale.P = 0.016), while a one-point increase in the ‘takes and stores food’ subscore was associated with a 0.72 unit increase in BMI, . No significant association was found between BMI and the ‘impairment of satiety’ sub-score.In a linear regression model adjusted for sex, a one-point score increase in the FRPQ ‘preoccupation with food’ subscore was associated with a 0.57 unit increase in BMI, , weight increased at an average rate of 0.08 kg/month . For these 41 participants, mean duration of weight trajectory data was 64.43 months (range 6–228); mean age at starting weight point was 28.8 ± 9.8 years; and mean age at terminal point was 34.4 ± 10.8 years. Compared with males, the rate of weight increase was greater in females by 0.10 kg/month .Across the subset of participants with available historic weight data (N =13); Group 2 ; and Group 3 . Individual subject’s slopes in Group 1 ranged from 0.11 to 0.60 (kg/month), while in Group 2 ranged from 0.02–0.09 (kg/month).Historic weight subset participants were classified into distinct weight trajectory groups  based onP = 0.778). Groups 1 and 2 were also not significantly different in average age at starting and terminal measurement . Group 1 scored higher on the FRPQ subscale ‘takes and stores food’ with a median score of 3.00 for Group 1 and 0.00 for Group 2, P = 0.003. Additional analyses revealed no significant differences between Groups 1 and 2 including: frequency of diabetes mellitus and use of levothyroxine; frequencies of reported weight-promoting medications (P = 0.339); percentages of individuals reporting first or second-degree family members with overweight/obesity (P = 0.547); or birth weight (g) (P = 0.487). Likewise, no differences were noticed between Group 1 and Group 2 in dietary intakes, or SCAS scores (see Supplementary Table 1).Groups 1 and 2 had a comparable percent of males  and food-related behaviors (based on FRPQ). Results from the DSQ revealed deviations from dietary recommendations for several components  in the WOn average, the WS cohort, as well as the NHANES controls, consumed less than the recommended intake of fruits, vegetables, whole grains, and dietary fiber, but greater than the allowance guidelines for added sugars , 15. ThiLow intake frequency of fruits and vegetables in WS, and lower plasma carotenoids compared with other IDD groups, has previously been described in a Norwegian cohort of adolescents and adults with WS . Plasma The mean daily intake of dietary fiber in our WS cohort was reduced by approximately 50% of the DGA recommendation . A commoAmong general population adults, numerous studies indicate that a poor-quality diet, one that is high in refined carbohydrates and low in fruits and vegetables, increases the risk of various medical problems including but not limited to obesity, diabetes mellitus, high blood pressure, and stroke –29. It iOne final result to expand on is that the daily calcium intake in the WS males exceeded the DGA recommendation and was also significantly higher than intakes for NHANES males and WS females. Since persons with WS are at an increased risk of hypercalcemia, especially during infancy, dietary calcium intake is often moderated by caregivers/families (most commonly as limiting dairy products)  and this2 to a high of 55.5 kg/m2 (median 27.3). The majority of the cohort (60%) was classified as having either overweight or obesity, but it is important to also point out that 11% of our study participants had a BMI in the underweight range (BMI ≤18.5).BMI among the WS adults in this study ranged from a low of 16.7 kg/mFour other WS series on comparably aged adults demonstrated a similar frequency of subjects with a BMI ≥ 25, ranging from 49 to 65% –11. In tBMI comparisons to other disorders with IDD is challenging due to methodologic differences and modest sample sizes. Excluding comparison to adults with Down syndrome, a group well-known to have a high prevalence of obesity, limited data suggests similar BMI distributions among adults with WS, adults with IDD of unknown etiology, and adults with autism spectrum disorder. However, being underweight may more commonly complicate the diagnosis of WS especially when compared with those with IDD of unknown etiology , 35. ChrWe are the first report on food-related behaviors, using the FRPQ, in adults with WS. Prevalent problematic food behaviors in WS  includedWe compared FRPQ findings from the WS cohort with other adults with IDD reported in the literature, namely: 1) adults with Prader–Willi syndrome (PWS); 2) adults with Smith–Magenis syndrome (SMS) using adult data kindly extracted by Dr. Sarah Elsea from Alaimo et al. ; and 3) The food-related behavior findings in WS suggest that the development of tailored weight reduction interventions for adults with WS might need to incorporate strategies on how to tackle and avoid some of these problematic behaviors. This perspective is supported by a small study in adolescents and young adults with Down syndrome. The largest weight loss occurred in the group where parents received education in behavioral strategies in addition to individual diet and exercise plans, while parents in the comparison group only received individual diet and exercise plans . BehavioA dearth of information on weight trajectories in adults with IDD exist and, to our knowledge, this study is the first to investigate individual trajectories. Those in Group 1  experienTo our knowledge, only a single previous study examined weight change over time in adults with IDD . Ptomey In our view, further investigation of adult weight trajectories is a rich area for future study as a means to identify biological, behavioral, or environmental factors that contribute to weight gain, that might prevent weight gain, or that cause and perpetuate being underweight. And though it was not a focus of our current analyses, patterns of high weight fluctuations occurred in several of our subjects [see unclassified group (Group 3) in Supplementary Fig. 1]. Scrutinizing the medical and psychological well-being of subjects at the time of these fluctuations may reveal predictable events, which could allow preventatives to be put in place to mitigate fluctuations.We elected to administer the DSQ based on several strengths of the instrument, including excellent validation in a large general population cohort by comparison to 24-h recall data  and simpIn terms of the FRPQ, it has been previously used in adults with PWS and SMS in multiple living situations , 17, 41.In terms of sample bias, it is possible that adults with WS and their families who have experienced issues with either low or excess body weight were more likely to take part in this study, which was described to families as focusing on factors contributing differences in body shapes and sizes. This bias does not invalidate the study’s findings but does limit their generalizability to the universe of adults with WS who may not have concerns about body shape.Lastly, analyses of weight trajectories were performed on relatively small samples sizes and were based on retrospectively collected medical records; therefore, data differed between subjects on variables such as ages for beginning and terminal weight points; duration of weight trajectory plots; time between measurements; and equipment used to obtain measurements. While a mixed model accounts for these factors, larger sample sizes and longer time intervals of data collection are needed to investigate weight trends and identify contributing dietary and non-dietary factors, including food behavior problems.Considerable variation in BMI was observed among adults with WS. Mean daily intakes of fruits, vegetables, and dietary fiber in WS adults, as well as in NHANES controls, were below national dietary guideline recommended ranges. Within our WS cohort, those with a BMI in either obesity or underweight category had decreased intake of fruits and vegetables compared with normal/overweight individuals while, additionally, individuals in the underweight category had decreased dietary fiber intake. These deviations from recommended intakes are an indicator that diet quality should be further evaluated in adults with WS.When assessing food behaviors, increased BMI and rate of weight gain were associated with increased preoccupation with food subscores and with a higher frequency of taking and storing food. For adults with WS and obesity or rapid weight gain, attention to problematic food behaviors may be an important aspect to consider and address.Click here for additional data file.


EAT thickness showed a significant correlation with GWI and GCW , and also independently correlated with GWI and GCW in the multivariate regression analysis . EAT thickness is associated with left ventricular myocardial function in subjects with suspected metabolic syndrome, independently of other risk factors. Further studies are supposed to ensure the causal associations and related mechanisms.Given the inconsistent results on the prognostic significance of epicardial adipose tissue (EAT), the purpose of the present study was to investigate the association of EAT thickness and myocardial work by non-invasive left ventricular pressure-strain loop in people with suspected metabolic syndrome (MS). A total of 194 participants imaged with echocardiography were evaluated. In accordance with the median EAT thickness, MS patients fell into thin EAT group and thick EAT group. Conventional echocardiographic parameters, global longitudinal strain (GLS) and the global myocardial work parameters obtained by pressure-strain loop analysis, comprising the global work index (GWI), global work efficiency (GWE), global constructive work (GCW) and global wasted work (GWW) were compared between the two groups. In comparison with the thin EAT group, thick EAT group achieved significantly higher values in interventricular septal thickness, end-diastolic left ventricular posterior wall thickness, left ventricular mass index and GWW ( EAT is considered to be an independent predictor of visceral obesity and may be closely associated with the pathogenesis of metabolic syndrome (MS)4, adverse clinical outcomes of COVID-197, coronary artery disease, and diabetes8.Epicardial adipose tissue (EAT) refers to a type of visceral fat located between the myocardial surface and the visceral layer of the pericardium. EAT has been confirmed as an independent risk factor for accelerated progression of subclinical coronary atherosclerosis9. Several cardiovascular risk factors occur together and are interrelated, with common potential mediators, mechanisms, and pathways10. MS turns out to be a leading health problem for its correlation with cardiovascular disease. It was reported to be correlated with a twofold increase in cardiovascular outcomes and a 1.5-fold increase in all-cause mortality11. It is clear why MS is the focus of various studies. The prevalence of MS has increased significantly worldwide, making accurate assessment of changes in left ventricular (LV) systolic function particularly important at an early stage. Tadic et al.12 evaluated left ventricular mechanics in patients with metabolic syndrome through two-dimensional echocardiography speckle tracking analysis, but the impact of metabolic syndrome on left ventricular myocardial work has not yet been evaluated.MS represents a group of common cardiovascular risk factors, comprising insulin resistance, obesity, atherosclerotic dyslipidemia, as well as hypertension13. Based on the speckle tracking echocardiography, brachial artery cuff pressure has been employed to replace left ventricular pressure, such that the LV strain and pressure parameters are combined, which can reduce the influence of afterload on the myocardial strain. Currently, limited data are available regarding the changes in left ventricular myocardium work and the correlation between epicardial fat and left ventricular myocardium work in MS patients.Ejection fraction (EF) is the most commonly used index of LV systolic function examined by conventional echocardiography. However, the measurement of EF is a simplistic method, limited by the requirements of geometric assumptions. Thus, in high-risk subclinical patients with MS, subtle changes may be overlooked when myocardial systolic function is assessed using EF alone. The use of two-dimensional speckle tracking echocardiography for myocardial deformation imaging has made the progress of left ventricular quantification beyond EF and become an excellent prognostic marker of cardiac events. However, one of the main limitations of strain imaging is load dependence, which may affect the diagnostic accuracy of myocardial function assessment. LV pressure-strain loop (PSL) analysis refers to a novel echocardiographic method for noninvasive quantification of myocardial workBased on the above, we hypothesized that the accumulation of EAT in patients with metabolic syndrome is related to sub-clinical changes in left ventricular myocardial work. Therefore, in this study, we aimed to investigate left ventricular systolic function and myocardial work in patients with suspected metabolic syndrome using left ventricular pressure-strain loop technique. Furthermore, we assessed the correlation between EAT thickness and left ventricular myocardial work in patients with suspected metabolic syndrome.14: Elevated triglycerides > 1.7 mmol/L; reduced high‐density lipoprotein cholesterol (HDL-C) (< 1.3 mmol/L in women and < 1.03 mmol/L in men); hypertension (HTN) (≥ 130/85 mmHg); hyperglycaemia ≥ 100 mg/dL; abdominal obesity by waist circumference ≥ 80 cm in women and ≥ 90 cm in men15. Furthermore, the exclusion criteria included participants with a previous history of coronary artery disease, congenital heart disease, heart failure, atrial fibrillation, moderate or severe valvular heart disease, stroke, or any revascularization. This study protocol gained approval from the Ethics Committee of the First Hospital of Qinhuangdao (2021Q082). The median EAT thickness reached 3.82 (Inter- Quartile Range 2.28) mm. In accordance with the median EAT thickness, MS patients were divided into two groups: thin EAT group  and thick EAT group . The clinical data, laboratory examination, echocardiography and myocardial work were compared between the two groups.Among 213 suspected MS patients imaged with echocardiography from September 2020 to December 2021, 194 patients were recruited. The exclusion criteria for the 19 patients comprised poor image quality (n = 12), and arrhythmia (n = 7). The median patient age was 62 years, 95 patients (49%) were men. The MS was defined based on the presence of three or more components of the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) criteria (2005 Revision)Anthropometry  was performed for all participants in the study to calculate body mass index (BMI). Moreover, waist circumference, heart rate, systolic (SBP) and diastolic blood pressures (DBP) were examined. Fasting plasma glucose (FPG), uric acid, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were examined less than two weeks prior to the echocardiographic evaluation using standard laboratory techniques.16. From the parasternal long axis view, we examined ascending aorta diameter (AAO), left atrial diameter (LAD), LV end-diastolic diameter (LVEDd), end-diastolic LV posterior wall (LVPWd) and interventricular septal (IVSd) thickness. LV ejection fraction (LVEF) was calculated using the modified Simpson's rule from apical 4- and 2-chamber views. LV mass (LVM) was determined using the Devereux formula17: LVM (g) = 0.8 × 1.04 × [(LVDd + IVS + LVPW)3 − LVDd3] + 0.6. The LV mass index (LVMI) was calculated by dividing the LVM by body surface area (BSA). BSA was calculated according to the Dubois formula18: BSA = 0.007184*Height0.725*Weight0.425. Pulse wave Doppler was adopted to measure the peak velocity of early and late mitral diastolic blood flow (E and A), tissue Doppler imaging was employed to measure the early diastolic motion velocity of mitral ring (e'), and E/e' were obtained.Echocardiographic examinations were performed using Vivid E95  ultrasound machine equipped with a M5S 3.5 MHz transducer (frame rate 50 ~ 80 frames/s). All records and measurements were taken according to American Association of Echocardiography guidelinesEAT refers to the fat pool between the outer wall of the myocardium and the visceral layer of the pericardium. The thickness of EAT was measured by echocardiography. Maximum EAT thickness was examined perpendicular to right ventricular free wall at the end of systole in a parasternal long-axis view. Three different measurements were recorded over three consecutive cardiac cycles, and the mean value of EAT was determined.GE EchoPAC version 203 software was employed for LV strain and myocardial work analysis. Three apical  views were captured and then stored for subsequent off-line analysis (average 3 consecutive cycles). Aortic valve closure was automatically recognized in apical long axis view. Click automated function imaging (AFI) button, the system automatically recognized the apical 4-chamber and 2-chamber view, and tracked the contour of the left ventricle endocardial and ventricular wall. The areas of interest were regulated by correcting endocardium boundaries or widths if required. The global longitudinal strain (GLS) was determined based on the weighted average of the peak systolic longitudinal strains at all 17 segments. After GLS was obtained, insertion brachial artery blood pressure, and timing of valve events, the software derived a non-invasive PSL. The global myocardial work parameters , global work efficiency (GWE), global constructive work (GCW), and global wasted work (GWW)) were obtained through the PSL analysis.p values ≤ 0.10 were included into the stepwise multiple regression analysis. A total of 20 patients were randomly selected and examined by two experienced cardiac sonographers. The consistency analysis of GWI, GCW, GWW and GWE was performed, and the same samples were analyzed again by one of the radiologists one week later. Intra-observer and inter-observer variables were calculated by intra-class correlation coefficient (ICC). A p value less than 0.05 was considered a difference that achieved statistical significance.The data were analyzed using SPSS software (version 27). Continuous variables are expressed as mean ± standard deviation if normally distributed or median with interquartile ranges if not normally distributed. Data are expressed as percentages in terms of categorical variables. Statistical comparisons comprised student t-test or Mann–Whitney U test. The correlation between different metabolic syndrome criteria and LV myocardial work parameters was determined using Pearson correlation coefficient. The variables with Informed consent was obtained from all subjects and/or their legal guardian(s). All methods were carried out in accordance with relevant guidelines and regulations. This study was approved by the Ethics Committee of the First Hospital of Qinhuangdao (2021Q082).p < 0.05; 138(10) vs. 140(20), p < 0.05; 80(12) vs. 85(12.25), p < 0.05; 93(12) vs. 97(15.75), p < 0.05; 2.01(1.08) vs. 2.59(2.74), p < 0.01). No significant differences were identified between the two groups in age, male proportion, heart rate, fasting blood glucose, uric acid, total cholesterol, LDL-C and HDL-C (p > 0.05).The baseline characteristics of MS patients are shown in Table p > 0.05). However, in comparison with the thin EAT group, thick EAT group showed significantly higher values in IVSd, LVPWd and LVMI (10(3) vs. 11(4), p < 0.05; 8(1) vs. 9(2), p < 0.001; 79.24(25.95) vs. 85.97(32.22), p < 0.05). The absolute value of GLS, E and e′ were significantly lower in the thick EAT group (17(2) vs.14(2.75), p < 0.001; 69.61(19.50) vs. 64(20.22), p < 0.05; 6.6(1.85) vs. 6(2.20), p < 0.01).Table p < 0.001). The GWI , GCW , and GWE (94(3) vs. 91(5); p < 0.001) were significantly decreased in the thick EAT group . Figure We further examined whether left ventricular myocardial work was affected by the change of EAT thickness. Detailed results on LV myocardial work are summarized in Table p = 0.001; r = − 0.253, p = 0.012) . Moreover, GCW showed a weak correlation with triglyceride . GWW and GWE had notable correlations with fasting blood glucose   Fig.  and SBP p = 0.001; β = − 0.199, p = 0.049) and SBP . GWW was independently correlated with fasting blood glucose   Compared with the thin EAT group, IVSd, LVPWd and LVMI in the thick EAT group were significantly increased, while the absolute values of GLS, E and e' were significantly decreased; (2) The thick EAT group achieved significantly higher value in GWW in comparison with the thin EAT group. Meanwhile, the GWI, GCW, and GWE were significantly decreased in the thick EAT group; and (3) EAT thickness was inversely correlated with GWI and GCW.19. In this study, conventional echocardiographic parameters were compared between the two groups. The results show that IVSd, LVPWd and LVMI in the thick EAT group were significantly increased compared with the thin EAT group, while the absolute values of GLS, E and e' were significantly decreased. In metabolic syndromes, left ventricular hypertrophy may occur with increased after-load20. It is the most prominent early cardiac manifestation in hypertensive patients, characterized by increased myocardial weight and ventricular remodeling. Diabetic myocardial microangiopathy can lead to chronic myocardial ischemia and hypoxia, cardiomyocyte necrosis, myocardial fibrocyte proliferation, and eventually left ventricular hypertrophy. Moreover, several inflammatory substances released from adipocytes, such as resistin have been suggested to contribute toward the adverse effects of obesity on the heart by promoting myocardial hypertrophy and dysfunction21. Extensive research has reported that the myocardial strain obtained by two-dimensional speckle tracking imaging can make left ventricular quantization beyond LVEF a better prognostic marker of cardiac events24. In this study, no significant difference in AAO, LAD, LVEDd, LVEF, A and E/e′ was reported between the thin and thick EAT groups. Compared with the traditional index of systolic function, GLS is more sensitive to indicate the degree of myocardial injury, and may decrease prior to the change of LVEF, consistent with previous literature reports. Among the measurements of myocardial function, GLS measured by echocardiography is the most widely studied marker, providing a simple, inexpensive, and quantitative way to assess global long-axis systolic function. However, one of the major limitations of strain imaging is after-load dependence. Moreover, it cannot reflect myocardial work or oxygen demand25. The noninvasive LV pressure-strain loop serves as a novel method of quantifying myocardial work in combination with myocardial strain and LV pressure measurements, and may provide incremental value for assessment of myocardial function26. The work contributed by the respective normal contracting component is positive and is called "constructive work". In the normal heart, very little work is wasted and thus there is a high work efficiency. Compared with the thin EAT group, GWW was significantly increased in thick EAT group, while GWI, GCW, and GWE notably declined. Prolonged systole and shortened myocardium after aortic valve closure will produce wasted work because it does not result in left ventricular ejection. In early untreated hypertension, diastolic dyssynchrony mainly affected constructive work, whereas post systolic shortening affected wasted work27.In general, the conventional clinical evaluation of left ventricular systolic function is performed through the measurement of myocardial fiber shortening indexes 28. However, excess body fat accumulation is correlated with metabolic abnormalities and myocardial dysfunction30. In this case, the protective properties of EAT may be destroyed and become harmful tissues that promote the development of cardiovascular diseases. Despite the unclear underlying mechanism, EAT has been confirmed as a potential source of inflammatory mediators, comprising interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Hirata et al. reported that the infiltration of inflammatory cells was enhanced in the epicardial adipose tissue of patients with coronary artery disease, instead of not in the subcutaneous fat31. Moreover, EAT has the potential to secrete adipotropic factors and free fatty acids, which may promote myocardial steatosis and induce cellular oxidative stress, as well as increased nitric oxide synthase activity and production of intracellular nitric oxide, and eventually lead to apoptosis of myofibrillar cells32. Recent research has placed a focus on myocardial fibrosis, a critical histological component of cardiac remodeling. Myocardial fibrosis scarring occurs most commonly after myocardial infarction, but can also occur in a variety of other diseases that promote myocardial fibrosis 33. As revealed by relevant data, EAT is capable of producing and secreting adipo-fibrokines , which may promote the formation of myocardial fibrosis34.To the best of our knowledge, this study has been the first to evaluate the role of MS on the correlation between EAT and myocardial work. The EAT is located outside the myocardial wall and maintains intimal contact with the epicardial vessels and myocardium, allowing for paracrine or vasocrine effects. Under health conditions, EAT has protective functions  while taking on critical significance in the energy supply of myocardium26. The left ventricular mechanics were significantly impaired by speckle tracking echocardiography in patients with metabolic syndrome. Previous studies have shown that epicardial fat volume is negatively correlated with left ventricular systolic dysfunction in patients with heart failure35. In this study, we found that EAT was negatively correlated with GWI and GCW in myocardial work parameters in patients with metabolic syndrome. In middle-aged male subjects with metabolic syndrome, EAT was correlated with inflammation represented by high‑sensitivity C‑reactive protein level, LV mass index, e′ and GLS, suggesting that the inflammatory activity of EAT induced myocardial remodeling and dysfunction29. Besides, this study further confirmed that EAT can have an adverse relationship with LV myocardial work at the early stage of LV remodeling.A significant correlation and agreement existed between the area of the non-invasive LV pressure-strain loop and glucose metabolism examined by positron emission tomography. Glucose metabolism is capable of indicating myocardial work. GCW refers to the work performed by the LV during contraction that contributes to LV ejection and is divided into shortening of cardiomyocytes during contraction and lengthening of cardiomyocytes during isovolumic relaxation. GWI is the total work in the LV pressure-strain loop area calculated from mitral valve closure to mitral valve opening36. Fibroblast growth factor-23 (FGF-23) is a biomarker of cardiovascular disease and may also be an early marker of cardiac injury in obese but otherwise healthy African American adolescents. Obesity may promote FGF-23 production in the absence of chronic kidney disease37. Previous studies have shown that blood pressure, waist circumference, and fasting glucose levels exert the most significant effect on left ventricular deformation of all metabolic syndrome components38. The findings in this study are similar to those of previous studies.Furthermore, LV myocardial work were also correlated with SBP, triglyceride, fasting blood glucose in metabolic syndrome. Men and women with impaired fasting glucose and impaired glucose tolerance have more severe alterations in cardiometabolic profiles and inflammatory markers than patients with impaired fasting glucose alone. Even in the absence of high blood pressure, these people were ten times more likely to develop preclinical cardiovascular diseaseSeveral limitations remained in this study. The correlation between increased EAT thickness and impaired LV myocardial work cannot prove that increased EAT inevitably leads to increased LV injury, and its pathophysiological mechanism needs to be further demonstrated. LV pressure-strain loop technology is based on two-dimensional speckle tracking echocardiography, which requires high-quality ultrasonic images. Brachial systolic pressure examined with a cuff and sphygmomanometer may be inaccurate because systolic pressure varies.Although no etiological relationships can be established from the current study, our intriguing finding of the independent relationship of EAT thickness and left ventricular myocardial function by LV pressure-strain loop in subjects with suspected metabolic syndrome may have important clinical implications. Moreover, EAT may serve as a useful marker for identifying metabolic syndrome patients at high risk of abnormal myocardial work. Nevertheless, there is no further mechanistic studies regarding associations between them. Further studies are supposed to ensure the causal associations and related mechanisms.