Objective:To establish and evaluate the performance of a stable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of six phytosterols in human plasma.Methods:The methodology was established, evaluated, and applied. A number of 136 healthy subjects were recruited from the Physical Examination Center of Suzhou Municipal Hospital from October 2023 to December 2023, meanwhile plasma phytosterols′ levels were validated in 5 patients with sitosterolemia. Taking deuterium isotope as internal standards, the clear separation was achieved using a Phenomenex C18 (2.1×100 mm, 2.6 μm) column. The mobile phase consisted of water and methanol at a flow rate of 0.4 ml/min with gradient elution, and the column temperature was 25 ℃. The experiments were performed on AB Sciex 6500+triple quadrupole mass spectrometry, and the linearity, limit of quantification, rate of recovery, precision, and stability of the method were evaluated. The testing method was further validated in the sitosterolemia patients.Results:The method had in-run and between-run coefficient of variation ranging 1.8%-12.5% and 2.6%-13.6%, respectively, which accorded with the precision requirements. The rates of recovery were 85.0%-105.1%, which accorded with the accuracy requirements. The samples showed good stability before and after handling stored at 4 ℃ for 72 hours. The plasma levels of sitosterol, campesterol, stigmasterol and cholestanol in sitosterolemia patients were apparently higher compared with those in the healthy group ( P<0.001). Conclusion:LC-MS/MS method for the quantitative measurement of plant sterols in human plasma has been established, which has convenient preprocessing and passed the performance evaluation including linearity, lower limit of quantification, recovery rate, precision and stability, and shown to be suitable for clinical application.

Objective:To quantitatively detect plasma ceramide (Cer) levels using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyze plasma ceramide levels in homozygous familial hypercholesterolemia (HoFH) patients.Methods:A case-control study was conducted. Plasma samples were collected from 70 healthy individuals who underwent physical examinations at the Health Check-Up Center of Suzhou Municipal Hospital between October 2023 and December 2023, as well as from 34 HoFH patients (including 11 patients with concomitant coronary artery disease) who were treated in the Cardiology Department of the same hospital during the same period. Deuterated isotopes were used as internal standards, and a Phenomenex Kinetex C18 column (2.1×100 mm, 2.6 μm) was employed for separation. An LC-MS/MS method was developed using an AB Sciex 6500+triple quadrupole mass spectrometer for the detection of ceramides, followed by method validation. Additionally, the differences in ceramide levels among healthy individuals, HoFH patients, and HoFH patients with concomitant coronary artery disease were compared. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of various ceramides for HoFH.Results:A method for the quantification of 9 plasma ceramides was established, showing excellent linearity ( r>0.99), precision (1.8%-11.0%), and recovery rates (89.9%-114.7%), meeting the requirements for detection. Compared to the control group, the HoFH group exhibited significantly higher levels of Cer(d18∶1/14∶0), Cer(d18∶1/16∶0), Cer (d18∶1/18∶0), Cer (d18∶1/18∶1), Cer (d18∶1/20∶0), Cer(d18∶1/22∶0), Cer(d18∶1/24∶0), and Cer(d18∶1/24∶1) ( P<0.05). ROC curve analysis revealed that Cer(d18∶1/18∶0) had the highest diagnostic value for HoFH, with an area under the curve (AUC) of 0.83, a cutoff value of 64.6 ng/ml, and sensitivity and specificity of 82.4% and 87.1%, respectively. In HoFH patients complicated with coronary artery disease, the plasma ceramide levels of Cer(d18∶1/18∶0) [(155.7±25.1) ng/ml vs (103.0±11.1) ng/ml, P=0.03] and the ratio of Cer (d18∶1/18∶0) to Cer(d18∶1/24∶0) (0.077±0.012 vs 0.048±0.006, P=0.03) were significantly higher than in HoFH patients without coronary artery disease. Conclusion:A quantitative LC-MS/MS method was developed for detecting ceramides in human plasma, which shows potential diagnostic value for patients with HoFH.

Objective:To establish and evaluate the performance of a stable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of six phytosterols in human plasma.Methods:The methodology was established, evaluated, and applied. A number of 136 healthy subjects were recruited from the Physical Examination Center of Suzhou Municipal Hospital from October 2023 to December 2023, meanwhile plasma phytosterols′ levels were validated in 5 patients with sitosterolemia. Taking deuterium isotope as internal standards, the clear separation was achieved using a Phenomenex C18 (2.1×100 mm, 2.6 μm) column. The mobile phase consisted of water and methanol at a flow rate of 0.4 ml/min with gradient elution, and the column temperature was 25 ℃. The experiments were performed on AB Sciex 6500+triple quadrupole mass spectrometry, and the linearity, limit of quantification, rate of recovery, precision, and stability of the method were evaluated. The testing method was further validated in the sitosterolemia patients.Results:The method had in-run and between-run coefficient of variation ranging 1.8%-12.5% and 2.6%-13.6%, respectively, which accorded with the precision requirements. The rates of recovery were 85.0%-105.1%, which accorded with the accuracy requirements. The samples showed good stability before and after handling stored at 4 ℃ for 72 hours. The plasma levels of sitosterol, campesterol, stigmasterol and cholestanol in sitosterolemia patients were apparently higher compared with those in the healthy group ( P<0.001). Conclusion:LC-MS/MS method for the quantitative measurement of plant sterols in human plasma has been established, which has convenient preprocessing and passed the performance evaluation including linearity, lower limit of quantification, recovery rate, precision and stability, and shown to be suitable for clinical application.

Objective:To quantitatively detect plasma ceramide (Cer) levels using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyze plasma ceramide levels in homozygous familial hypercholesterolemia (HoFH) patients.Methods:A case-control study was conducted. Plasma samples were collected from 70 healthy individuals who underwent physical examinations at the Health Check-Up Center of Suzhou Municipal Hospital between October 2023 and December 2023, as well as from 34 HoFH patients (including 11 patients with concomitant coronary artery disease) who were treated in the Cardiology Department of the same hospital during the same period. Deuterated isotopes were used as internal standards, and a Phenomenex Kinetex C18 column (2.1×100 mm, 2.6 μm) was employed for separation. An LC-MS/MS method was developed using an AB Sciex 6500+triple quadrupole mass spectrometer for the detection of ceramides, followed by method validation. Additionally, the differences in ceramide levels among healthy individuals, HoFH patients, and HoFH patients with concomitant coronary artery disease were compared. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of various ceramides for HoFH.Results:A method for the quantification of 9 plasma ceramides was established, showing excellent linearity ( r>0.99), precision (1.8%-11.0%), and recovery rates (89.9%-114.7%), meeting the requirements for detection. Compared to the control group, the HoFH group exhibited significantly higher levels of Cer(d18∶1/14∶0), Cer(d18∶1/16∶0), Cer (d18∶1/18∶0), Cer (d18∶1/18∶1), Cer (d18∶1/20∶0), Cer(d18∶1/22∶0), Cer(d18∶1/24∶0), and Cer(d18∶1/24∶1) ( P<0.05). ROC curve analysis revealed that Cer(d18∶1/18∶0) had the highest diagnostic value for HoFH, with an area under the curve (AUC) of 0.83, a cutoff value of 64.6 ng/ml, and sensitivity and specificity of 82.4% and 87.1%, respectively. In HoFH patients complicated with coronary artery disease, the plasma ceramide levels of Cer(d18∶1/18∶0) [(155.7±25.1) ng/ml vs (103.0±11.1) ng/ml, P=0.03] and the ratio of Cer (d18∶1/18∶0) to Cer(d18∶1/24∶0) (0.077±0.012 vs 0.048±0.006, P=0.03) were significantly higher than in HoFH patients without coronary artery disease. Conclusion:A quantitative LC-MS/MS method was developed for detecting ceramides in human plasma, which shows potential diagnostic value for patients with HoFH.

Myasthenia gravis (MG) is an autoimmune disease of the nervous system mediated by autoimmune antibodies, dependent on T cells and involved in multiple complement. Recent years, targeted biologics have shown advantages in a number of clinical studies of myasthenia gravis. This review focuses on targeted therapy on B cells, complement, neonatal fragment crystal receptor (FcRn) and cytokine monoclonal antibodies, as well as on the latest research progress of chimeric antigen receptor T cells (CAR-T) or chimeric autoantibody receptor T cells (CAAR-T) in MG therapy, in order to provide the latest drug information for clinicians.

Objective:To analyze the clinical features and genetic etiology of 17 Chinese pedigrees affected with X-linked intellectual disability (XLID).Methods:Seventeen pedigrees affected with unexplained intellectual disability which had presented at Henan Provincial People′s Hospital from May 2021 to May 2023 were selected as the study subjects. Clinical data of the probands and their pedigree members were collected. Trio-whole exome sequencing (Trio-WES), Sanger sequencing and X chromosome inactivation (XCI) analysis were carried out. Pathogenicity of candidate variants was predicted based on the guidelines from the American College of Medical Genetics and Genomics and co-segregation analysis.Results:The 17 probands, including 9 males and 8 females with an age ranging from 0.6 to 8 years old, had all shown mental retardation and developmental delay. Fourteen variants were detected by genetic testing, which included 4 pathogenic variants ( MECP2: c. 502C>T, MECP2: c. 916C>T/c.806delG, IQSEC2: c.1417G>T), 4 likely pathogenic variants ( MECP2: c. 1157_1197del/c.925C>T, KDM5C: c. 2128A>T, SLC6A8: c. 1631C>T) and 6 variants of uncertain significance ( KLHL15: c. 26G>C, PAK3: c. 970A>G/c.1520G>A, GRIA3: c. 2153C>G, TAF1: c. 2233T>G, HUWE1: c. 10301T>A). The PAK3: c.970A>G, GRIA3: c. 2153C>G and TAF1: c. 2233T>G variants were considered as the genetic etiology for pedigrees 12, 14 and 15 by co-segregation analysis, respectively. The proband of pedigree 13 was found to have non-random XCI (81: 19). Therefore, the PAK3: c. 1520G>A variant may underlie its pathogenesis. Conclusion:Trio-WES has attained genetic diagnosis for the 17 XLID pedigrees. Sanger sequencing and XCI assay can provide auxiliary tests for the diagnosis of XLID.

Objective:To evaluate the predictive value of stress+ rest gated myocardial perfusion imaging (G-MPI) in assessing all-cause mortality risk in patients with familial hypercholesterolemia (FH).Methods:From June 2010 to March 2022, 72 patients (39 males, 33 females; age (21.1±12.3) years) who diagnosed with FH clinically and genetically and underwent stress+ rest G-MPI in Beijing Anzhen Hospital, Capital Medical University were retrospectively followed up. Image analysis was performed using the 17-segment 5-point method to obtain left ventricular myocardial perfusion and functional parameters. Patients were followed for all-cause mortality events, and predictors associated with the risk of all-cause mortality were analyzed using Cox regression. The efficiencies of predictors were evaluated by ROC curve analysis, and the Kaplan-Meier method and log-rank test were used to compare the differences in the incidence of all-cause mortality in different groups of patients with FH. Independent-sample t test or Mann-Whitney U test was used to analyze the data. Results:The follow-up time of 72 patients was 7(4, 10) years, and all-cause death occurred in 16(22.2%) patients during the follow-up period. There were statistically significant differences in total cholesterol (TC), low density lipoprotein cholesterol (LDLC), summed stress score (SSS), summed rest score (SRS), summed difference score (SDS), stress end-systolic volume (SESV), stress ejection fraction (SEF), rest end-diastolic volume (REDV), rest end-systolic volume (RESV) and rest ejection fraction (REF) between the death group and the survival group ( t values: from -2.65 to 4.47, z values: from -3.43 to -1.98, all P<0.05). Cox regression analysis showed that SDS (hazard ratio ( HR)=1.337, 95% CI: 1.114-1.604, P=0.002), SESV ( HR=1.019, 95% CI: 1.008-1.030, P<0.001) and LDLC ( HR=1.355, 95% CI: 1.049-1.749, P=0.020) were independent predictors associated with the risk of all-cause mortality in patients with FH. The optimal cut-off value of SESV for predicting mortality in patients with FH determined by ROC curve analysis was 35.5 ml, with the AUC of 0.701 (95% CI: 0.517-0.885). The incidence of all-cause mortality in the group with SESV≥35.5 ml was significantly higher than that in the group with SESV<35.5 ml (28.6% vs 6.9%; χ2=5.15, P=0.023). Conclusion:Stress+ rest G-MPI is an important imaging method for all-cause mortality risk assessment in patients with FH, and SDS, SESV and LDLC are important factors in predicting mortality in patients with FH.

Phytosterolemia is a rare, severe autosomal recessive sterol storage disorder caused by homozygous or compound heterozygous mutations in one of the ABCG5 and/or ABCG8 adenosine triphosphate binding cassette (ABC) genes. The most prominent features of phytosterolemia are the significantly increased serum content of plant sterols. Present review focused on the laboratory diagnosis of phytosterolemia, briefly described the metabolism of phytosterols, and introduced the latest research progress on phytosterolemia diagnosis, its relationship with ASCVD and laboratory diagnostic methods (including the detection of serum concentrations of phytosterols, ABCG5/G8 gene mutation). We hope this article could improve readers′ awareness and attention on this disease.

Plant sterols are a class of compounds naturally produced by plants and structurally similar to cholesterol. For human, plant sterols can only be obtained from food and cannot be utilized directly. With the development of gas chromatography technology and in-depth research on the molecular mechanisms of rare diseases, plant sterols have been found to play important roles in atherosclerotic cardiovascular disease (ASCVD)and become a residual risk factor after statin therapy. As a novel metabolic marker, plant sterols can reflect steady state the imbalance of the cholesterol and the increase of the cholesterol absorption within the human body, and is expected to become a new risk factor for ASCVD residual risk assessment.

Objective To evaluate the features of myocardial perfusion imaging (MPI) in patients with homozygous familial hypercholesterolemia (HoFH) and its influence factors.Methods Forty-two consecutive HoFH patients (21 males,21 females;average age:(14.8±8.4) years) were retrospectively enrolled in this study from June 2010 to November 2016.Diagnosis was proved by clinical and chromosome tests,and all patients underwent ATP stress and rest 99Tcm-methoxyisobutylisonitrile (MIBI) SPECT MPI with a two-day protocol.Summed stress score (SSS) and summed rest score (SRS) were acquired,and summed difference score (SDS;SSS-SRS) was calculated.Relations between SSS,SRS,SDS and age,lipid profile were analyzed.Two-sample t test,x2 test,multiple linear regression analysis and multivariate logistic regression were used to analyze the data.Results There were 24 patients with positive MPI results (SSS≥1),and females (76.2％,16/21) showed more positive MPI results than males (38.1％,8/21;x2=6.22,P＜0.05).Eighteen patients had negative MPI results.There were 6,8,10 patients with MPI positive results in ＜ 10 years group (n =14),10-18 years group (n =14) and ≥ 19 years group,respectively (x2=2.33,P＞0.05).Positive electrocardiograph (ECG) in ATP stress test was observed in 9 females (42.9％,9/21) and 3 males (14.3％,3/21;x2 =4.20,P＜0.05).Sixty-three (8.8％,63/714) abnormal myocardial perfusion segments (SSS≥ 1) were found,which was mainly (60.3％,38/63) distributed in myocardial regions supplied by left anterior descending branch (LAD).SSS was positively correlated with age and high density lipoprotein cholesterol (HDLC).SRS,SDS were positively correlated with HDLC and age respectively.Multivariate logistic regression analysis indicated that the female was the only independent risk factor to predict positive MPI (odds ratio=5.2,95％ CI:1.363-19.774).Conclusions In HoFH patients,abnormal myocardial perfusion had a rising trend with age increasing.Female patients are more likely to have abnormal MPI.Abnormal myocardial perfusion segments are mainly located in myocardial regions supplied by LAD.Age and gender are influence factors of abnormal MPI in HoFH patients.