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The aim of this study is to investigate the status of laboratory practice of organic acid (OA) analysis using gas chromatography–mass spectrometry in China.

This study aims to evaluate the commutability of external quality assessment (EQA) materials and candidate reference materials (RMs) for plasma renin activity (PRA) assay. Commutabilities of 16 candidate RMs were measured along with 40 clinical samples by the four different routine PRA assays, including three LC‒MS/MS assays and one chemiluminescence immunoassay. Sixteen candidate RMs included native/spiked human plasma pools (small-scale pools with <50 individuals) and current EQA materials (large-scale pools with >1,000 individuals). Difference in bias approach and linear regression with prediction interval approach were adopted to determine the commutability. Two-way variance analysis was used to estimate the effects of spiked and pool size on the commutability. Stability and homogeneity studies were performed. Precision and correlation performance of all assays was acceptable. In the difference in bias approach, the commutability results were not satisfactory (noncommutability: 14/16) and significant sample-specific effects were detected in assay pairs using different incubation buffers. For the prediction interval approach, no commutability was observed in the spiked small-scale pools; EQA materials (4/9) had more satisfactory commutability among all assays than the small-scale pools (2/7); RMs of large-scale pools tend to have better commutability no matter spiked or not. Commutable RMs were obtainable but challenging. Current EQA materials with relatively good commutability, stability, and homogeneity were appropriate RMs. Large-scale pools are tending to be commutable. Spiking in small-scale pools was not suggested to prepare RMs. MPs adopting a uniform incubation buffer would be preferable for further commutability research. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia is a risk factor for various cardiovascular diseases. However, the mechanism underlying homocysteine- (Hcy-) induced vascular injury remains unclear. The purpose of the present study was to examine a potential mechanism by which Hcy induced injury in human umbilical vascular endothelial cells (HUVEC). The protein abundance of autophagy-related markers was markedly decreased after Hcy treatment, which was associated with endoplasmic reticulum (ER) stress and apoptosis in HUVECs. Protein expression level of angiotensin II type 1 receptor (AT1 receptor) was dramatically increased in response to Hcy. Valsartan, an AT1 receptor blocker, improved autophagy and prevented ER stress and apoptosis in HUVECs treated with Hcy. Consistent with this, silence of AT1 receptor with siRNA decreased the protein abundance of ER stress markers, prevented apoptosis, and promoted autophagy in HUVECs. Inhibition or knockdown of AT1 receptor was shown to be associated with suppression of p-GSK3β/GSK3β-p-mTOR/mTOR signaling pathway. Additionally, inhibition of autophagy by 3-MA aggravated Hcy-induced apoptosis, while amelioration of ER stress by 4-PBA prevented Hcy-induced injury in HUVECs. Hcy-induced HUVEC injury was likely attributed to AT1 receptor activation, leading to impaired autophagy, ER stress, and apoptosis. [ABSTRACT FROM AUTHOR]

This study aims to investigate and update the consistency and comparability of plasma renin activity (PRA) assays in measuring clinical samples. The contributions of recalibration, blank subtraction, and incubation strategies to interchangeability were also explored. Five different laboratories were evaluated using forty-six individual plasma samples, including four liquid chromatography-tandem mass spectrometry (LC‒MS/MS) assays and one chemiluminescence immunoassay (CLIA). Spearman correlation coefficient (R), Passing–Bablok regression, and Bland‒Altman plot analyses were used to evaluate the consistency among assays. Consistency before and after recalibration, blank subtraction, and incubation strategy unification was compared. A good correlation was observed among all assays (R>0.93). None of the samples measured by all assays showed coefficient variation (CV) <10 %, and 37 % of samples showed overall CVs >20 %. The 95 % confidence intervals (CIs) for slopes did not contain 1 for most assay pairs. Large relative biases (−85.1–104.2 %) were found, and 76 % (52–93 %) of samples had unacceptable biases. Recalibration reduced the calibration bias. Ignoring blank subtraction improved the comparability across all assays while unifying incubation did not. The interchangeability of PRA measurement was unsatisfying. Harmonization on calibrator and ignoring blank were recommended. Unifying incubation strategy was unnecessary. [ABSTRACT FROM AUTHOR]

• LC-MS/MS assay-specific RIs were established for serum folates vitamers. • The potential over-usage of FA supplements was observed in folates deficiency patients. • The LC-MS/MS assay-specific folates RIs for the evaluation of individual nutrition conditions and over-dosage monitoring were recommended. Little known about folates status in folate deficiency patients. This study aims to establish liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay-specific reference intervals (RIs) for serum 5-Methyltetrahydrofolate (5MeTHF), folic acid (FA), 5-Formyltetrahydrofolate (5FoTHF), and total folate (TFOL), and investigate the folates status in FA-supplemented folate deficient patients. Sera from 120 reference subjects were selected and measured. An LC-MS/MS method for serum 5MeTHF, FA, and 5FoTHF was employed. RIs were derived based on the CLSI C28-A3. Serum folate levels of 38 FA-supplemented folate deficiency patients were analyzed. RIs (median) for 5MeTHF, FA, 5FoTHF, and TFOL were 3.83–62.33 nmol/L (12.27 nmol/L), 0.30–0.92 nmol/L (0.49 nmol/L), <0.73 nmol/L (0.00 nmol/L), and 4.17–63.47 nmol/L (12.66 nmol/L), respectively. Approximately 53 % (20/38), 74 % (28/38), and 63 % (24/38) of patients presented high levels of 5MeTHF, FA, and TFOL, respectively, which far exceeded the upper reference limit (URL) of the corresponding RIs. A half (18/38) of patients showed simultaneously higher 5MeTHF and FA levels which were beyond the URLs of RIs. Near one-third (11/38) of patients exhibited extremely high FA levels which exceeded the 100-fold URL of RIs. The highest levels can be 539 nmol/L for FA, 364 nmol/L for 5MeTHF, and 686 nmol/L for TFOL. LC-MS/MS assay-specific RIs were established for folates vitamers without age or gender partitioning. Abnormally high levels of unmetabolized FA and 5MeTHF were observed in quite a few FA-supplemented patients. Considering the adverse risks caused by folates excess, we appeal for a justifiable and individualized FA supplementation. We also recommend establishing LC-MS/MS assay-specific RIs for routine monitoring of folates status. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? An established method for trace, simple, rapid, cheap, sensitive, accurate, robust, and high-throughput for methylmalonic acid quantification. Good chromatographic separation of MMA and its intrinsic isomer and good signals of MMA were achieved using a simple isocratic elution strategy. Materials and reagents that are complex or not always accessible and procedures in previous methods such as derivatization, multistep SPE, incubations, evaporations, drying, or reconstitutions are not required in this MMA quantification method. What is the implication of the main finding? This method is suitable for large-scale MMA testing. Background: Methylmalonic acid (MMA) is an essential indicator of vitamin B12 (VB12) deficiency and inherited metabolic disorders (IMDs). The increasing number of requests for MMA testing call for higher requirements for convenient MMA testing methods. This study aims to develop a convenient quantification method for serum MMA. Methods: The method was established based on the stable isotope-dilution liquid chromatography–tandem mass spectroscopy (ID-LC-MS/MS) technique. The LC-MS/MS parameters and sample preparation were optimized. Specificity, sensitivity, robustness, accuracy, and clinical applicability were validated according to CLSI C62-A guidelines. MMA levels in VB12-sufficient subjects and VB12-deficient subjects were measured. Results: MMA and its intrinsic isomer, i.e., succinic acid (SA), were completely separated. The average slope, intercept, and correlation relationship (R) with 95% confidence intervals, during the two months, were 0.992 (0.926–1.059), −0.004 (−0.012–0.004), and 0.997 (0.995–0.999), respectively. The limit of detection and quantification were <0.058 μmol/L and 0.085 μmol/L, respectively. Intra-run, inter-run, and total imprecisions were 1.42–2.69%, 3.09–5.27%, and 3.22–5.47%, respectively. The mean spiked recoveries at the three levels were 101.51%, 92.40%, and 105.95%, respectively. The IS-corrected matrix effects were small. The VB12-deficient subjects showed higher MMA levels than VB12-sufficient subjects. Conclusions: A convenient LC-MS/MS method for serum MMA measurement was developed and validated, which could be suitable for large-scale MMA testing and evaluating MMA levels in VB12-deficient patients. [ABSTRACT FROM AUTHOR]

Accurate measurement of serum folate is essential for the diagnosis and management of various disorders. This study aims to investigate the between-method differences of four immunoassays and a rapid isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method. Roche Cobas (USA), Abbott Alinity i2000 (USA), Beckman Coulter Access (USA), Mindray CL-6000i (China), and the ID-LC-MS/MS method were compared using 46 human serum samples. The results were analysed by Passing–Bablok regressions and Bland–Altman plots. A bias of 13.31% based on biological variation was used as the bias criterion. All the within-run and total coefficients of variation (CVs) met the specification. The folate concentrations determined by all the assays were significantly different (p=0.0028). All assays had correlation coefficients over 0.97 with each other. The 95% confidence intervals (CIs) for the slope seldom contained 1 and few 95% CIs for the intercept contained 0 in the regression equations. Compared to ID-LC-MS/MS, the biases of all assays ranged from −20.91 to 13.56 nmol/L, and the mean relative biases ranged from −9.85 to 40.33%. The predicted mean relative biases at the medical decision levels rarely met the criterion. Assays for serum folate had good correlations with each other but lacked good agreement. The accuracy and consistency of assays for serum folate should be measured and assessed routinely. Standardization work to improve the accuracy of serum folate assays, such as the extension of traceability to reference methods or materials, calibration standardization efforts, and assay-adjusted cut-offs should be promoted. [ABSTRACT FROM AUTHOR]

Background: We examined the genetic background of a Chinese Han family in which some members presented with complex arrhythmias including sick sinus syndrome, progressive conduction block, atrial fibrillation, atrial standstill and Brugada syndrome. The possible underlying mechanism associated with the genetic mutation was explored. Methods: Targeted capture sequencing was conducted in the probands in the coding and splicing regions of genes implicated in inherited arrhythmias. Stable cell lines overexpressing wild type (WT) or mutant SCN5A were generated in HEK293T cells. Whole‐cell recording was performed to evaluate the functional changes in sodium channels. Results: The rare heterozygous linkage mutations, SCN5A R965C and R1309H, were found in these patients with complex familial arrhythmias. Compared to WT, R965C or R1309H, the peak current of sodium channel was dramatically reduced in HEK293T cell with linkage R965C‐R1309H mutation when testing potentials ranging from −45 to 15 mV. Notably, the maximum peak current of sodium channels with R1309H and linkage R965C‐R1309H displayed significant decreases of 31.5% and 73.34%, respectively, compared to WT. Additionally, compared to R965C or R1309H alone, the linkage mutation R965C‐R1309H demonstrated not only a more obvious depolarisation‐shifted activation and hyperpolarisation‐shifted inactivation, but also a more significant alteration in the time constant, V1/2 and the slope factor of activation and inactivation. Conclusions: The linkage mutation SCN5A R965C‐R1309H led to a more dramatically reduced current density, as well as more significant depolarisation‐shifted activation and hyperpolarisation‐shifted inactivation in sodium channels than R965C or R1309H alone, which potentially explain this complex familial arrhythmia syndrome. [ABSTRACT FROM AUTHOR]

Commutability assessment, commutability criterion, difference in bias approach, sample specific effects, Deming regression The Deming analysis approach [[1]] has been the golden standard for commutability assessment for years [], but the commutability criterion [[1]], i.e., the width of the prediction interval, is easily affected by the presence of large sample-specific effects and varies among different method comparisons []. Keywords: commutability assessment; commutability criterion; Deming regression; difference in bias approach; sample specific effects EN commutability assessment commutability criterion Deming regression difference in bias approach sample specific effects e235 e236 2 08/30/22 20220901 NES 220901 To the Editor, Commutability is an important characteristic for reference materials used in standardization activities. [Extracted from the article]

Calcium ion is an important cation influencing the binding of recalcitrant organic contaminants with activated sludge during wastewater treatment process, but there is still unknown about its role in amphoteric fluoroquinolones binding. Binding experiments show that Ca2+ markedly inhibited binding of ciprofloxacin (CIP) onto sludge, causing 7–203 times of CIP release. Multi-spectroscopic examinations indicate that tryptophan-like and tyrosine-like proteins in extracellular polymeric substances (EPS) were dominant components for CIP binding by static quenching and forming CIP-proteins complexes. Addition of Ca2+ into EPS and CIP binding systems induced increase of association constants (from 0.024–0.064 to 0.027–0.084 L/μmol) and binding constants (from 0.002–0.039 to 0.012–0.107) and decrease of binding sites number (from 0.893–2.007 to 0.721–1.386). Functional groups of EPS and secondary structure of proteins were remarkably changed upon reactions with CIP and Ca2+. Calcium ion interacted with EPS and CIP binding system in two distinct ways: Ca2+ shielded C O in amide I in EPS for CIP binding, whereas strengthened binding between CIP and functional groups including C O in carboxyl groups in extra-microcolony polymers and O H in extra-cellular polymers by forming ternary complexes. Cation competition for C O in amide I is responsible for Ca2+ induced CIP release from the sludge. Results suggest the highly potential release of CIP from high saline wastewater and cation-conditioned sludge which needs further monitoring and evaluation. Unlabelled Image • Ca2+ markedly inhibited binding of ciprofloxacin to sludge. • Proteins in EPS are critical in providing binding sites for ciprofloxacin and Ca2+. • Ca2+ acted mainly through complexation and cation competition. • Ternary complex formed among Ca2+, ciprofloxacin, and C=O in carboxyl group and O–H • C=O in amide I is key group for Ca2+ competition inducing ciprofloxacin release. [ABSTRACT FROM AUTHOR]

Objectives: This study aims to evaluate the commutability of external quality assessment (EQA) materials and candidate reference materials (RMs) for plasma renin activity (PRA) assay., Methods: Commutabilities of 16 candidate RMs were measured along with 40 clinical samples by the four different routine PRA assays, including three LC‒MS/MS assays and one chemiluminescence immunoassay. Sixteen candidate RMs included native/spiked human plasma pools (small-scale pools with <50 individuals) and current EQA materials (large-scale pools with >1,000 individuals). Difference in bias approach and linear regression with prediction interval approach were adopted to determine the commutability. Two-way variance analysis was used to estimate the effects of spiked and pool size on the commutability. Stability and homogeneity studies were performed., Results: Precision and correlation performance of all assays was acceptable. In the difference in bias approach, the commutability results were not satisfactory (noncommutability: 14/16) and significant sample-specific effects were detected in assay pairs using different incubation buffers. For the prediction interval approach, no commutability was observed in the spiked small-scale pools; EQA materials (4/9) had more satisfactory commutability among all assays than the small-scale pools (2/7); RMs of large-scale pools tend to have better commutability no matter spiked or not., Conclusions: Commutable RMs were obtainable but challenging. Current EQA materials with relatively good commutability, stability, and homogeneity were appropriate RMs. Large-scale pools are tending to be commutable. Spiking in small-scale pools was not suggested to prepare RMs. MPs adopting a uniform incubation buffer would be preferable for further commutability research., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Importance: Knowing the scale of rare inborn errors is important for screening and resource allocation. Evidence on the prevalence of methylmalonic acidemia (MMA) among newborns and the clinical-suspected population from large-scale screening programs needs to be systematically synthesized., Objective: To estimate the worldwide prevalence of MMA for newborns and the clinical-suspected population and explore the differences in different regions, periods, and diagnostic technologies., Data Sources: MEDLINE, Embase, CRD, Cochrane Library, Scopus, CINAHL, and PROSPERO. Study Selection: All studies reporting the epidemiology characteristics of MMA were selected., Data Extraction and Synthesis: Characteristics of study, subjects, and epidemiology were extracted, random-effect models were used for meta-analyses., Main Outcome and Measure: Pooled prevalence of MMA., Results: This study included 111 studies. The pooled prevalence of MMA worldwide was 1.14 per 100,000 newborns (1516/190,229,777 newborns, 95% CI: 0.99-1.29) and 652.11 per 100,000 clinical-suspected patients (1360/4,805,665 clinical-suspected individuals, CI: 544.14-760.07). Asia and Africa got a higher pooled prevalence of MMA. The prevalence of MMA in newborns increased through the years, while that in the clinical-suspected population decreased. Collecting blood ≥ 72 h after birth had a higher pooled prevalence of MMA than collecting during 24 h-72 h after birth. The combining-use of MS/MS and GC/MS had a higher pooled prevalence than the single-use of MS/MS or GC/MS. Prevalence of cbl C, mut, cbl B, cbl A, isolated MMA, combined MMA and homocystinuria, vitamin B12-responsive MMA was synthesized., Conclusions and Relevance: Prevalence of MMA among newborns was extremely low, but considerably high in the clinical-suspected population, indicating the need for more efficient newborn screening strategies and closer monitoring of the high-risk population for the early signs of MMA. Asia and Africa should attach importance to the high prevalence of MMA. Further diagnostic tests were recommended for the combining-use vs single-use of MS/MS and GC/MS and for collecting blood after 72 h vs during 24-72 h after birth.

Background: Coronavirus disease 2019 (COVID-19) has already became a public health emergency of international concern. COVID-19 related cardiac injury remains largely unclear., Methods: We retrospectively analyzed demographic, clinical, laboratory and cardiovascular imaging data of all consecutively admitted adult COVID-19 patients in Zhuhai, China from January 17th, 2020 to February 18th, 2020., Results: A total of 93 patients were included in the study. Acute cardiac injury was found in 9 (9.7%) COVID-19 patients with median level of hypersensitive cardiac troponin I (hs-cTnI) to be 0.085 µg/L (IQR 0.027-0.560 µg/L). Compared with patients without cardiac injury, the median age of patients with cardiac injury was significantly older (65.0 vs. 44.0, P<0.05), hypertension was significantly more common (44.4% vs. 14.3%, P<0.05), and the proportion of severe-critical cases were greater (77.8% vs. 17.9%, P<0.05). Patients with cardiac injury were more likely have elevation of N-terminal proBNP (NT-proBNP) in comparison (66.7% vs. 10.0%, P<0.05). There was no significant difference in echocardiographic parameters between patients with and without cardiac injury. Multivariable logistic regression analysis indicated that older age (OR: 1.093, 95% CI: 1.011-1.182) and increased NT-proBNP (OR: 10.979, 95% CI: 2.024-59.555) were independent risk factors for cardiac injury. Cardiac magnetic resonance (CMR) imaging performed on three patients at around one month after they underwent significant hs-cTnI elevation showed that they had underlying cardiovascular comorbidities., Conclusions: Acute cardiac injury was seen in the minority of hospitalized COVID-19 patients in Zhuhai, China. Older age and increased NT-proBNP were associated with acute cardiac injury., Registration Number: ChiCTR2000030952., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/cdt-20-607). All authors have no conflicts of interest to declare., (2020 Cardiovascular Diagnosis and Therapy. All rights reserved.)

Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. However, it is unclear whether Ku80 regulates tumor growth in human melanoma. In this study, we screened a siRNA library targeting 6024 human genes and identified Ku80 as a potential therapeutic target in melanoma cells. Knockdown of Ku80 significantly suppressed melanoma cell proliferation and induced apoptosis, as well as enhanced the antitumor effect of melatonin in melanoma in vitro and in vivo. Overexpression of Ku80, however, promoted melanoma growth and increased the insensitivity of melanoma cells to melatonin. Mechanistically, we found that Ku80 bound to the PDK1 promoter and activated the transcription of PDK1. Moreover, we showed that the binding of Ku80 at the PDK-1 promoter was HIF1-α dependent, and melatonin degraded HIF1-α in melanoma cells. Furthermore, clinical data revealed that the expression of Ku80 and PDK-1 proteins were positively correlated and elevated in the tumor tissues of melanoma patients, and high expression of Ku80 predicted a poor prognosis in melanoma. Collectively, our study demonstrated that Ku80 promoted melanoma growth and regulated antitumor activity of melatonin by targeting HIF1-α dependent PDK-1 signaling pathway, suggesting that Ku80 may be a potential molecular target for melanoma treatment., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The molecular mechanisms of melamine-induced renal toxicity have not been fully understood. The purpose of the study aimed to investigate whether melamine and cyanuric acid induced NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in the kidney, which may contribute to abnormal water and sodium handling in a rat model. Wistar rats received melamine (Mel; 200 mg·kg body wt -1 ·day -1 ), cyanuric acid (CA; 200 mg·kg body wt -1 ·day -1 ), or Mel plus CA (Mel + CA; 100 mg·kg body wt -1 ·day -1 , each) for 2 wk. Mel + CA caused damaged tubular epithelial structure and organelles, dilated tubular lumen, and inflammatory responses. Crystals were observed in urine and serum specimen, also in the lumen of dilated distal renal tubules. The combined ingestion of Mel and CA in rats caused a markedly impaired urinary concentration, which was associated with reduced protein expression of aquaporin (AQP)1, 2, and 3 in inner medulla and α-Na-K-ATPase and Na-K-2Cl transporters in cortex and outer medulla. Mel + CA treatment was associated with increased protein expression of CD3 and mRNA levels of CD68 and F4/80 as well as phosphorylation of NF-κB in the kidney. Mel + CA treatment increased protein and mRNA expression of NLRP3 inflammasome components apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, and IL-1β in the inner medulla of rats. NF-κB inhibitor Bay 11-7082 reduced IL-1β expression induced by Mel + CA and prevented downregulation of AQP2 in inner medullary collecting duct cell suspensions. In conclusion, Mel + CA treatment caused urinary-concentrating defects and reduced expression of renal AQPs and key sodium transporters, which is likely due to the inflammatory responses and activation of NLRP3 inflammasome induced by crystals formed in the kidney.

Background: MicroRNAs (miRNAs) have been found to be involved in coronary artery disease (CAD) and corresponding disease severity. The aim of this study is to determine plasma levels of miR-144 in CAD and its association with the severity of this disease., Methods: Plasma levels of miR-144 in 60 CAD patients including stable angina pectoris (SAP) (n=29), unstable angina pectoris (UAP) or non-ST elevation myocardial infarction (MI) (NSTEMI) (n=17), or ST-elevation MI (STEMI) (n=14) and 20 non-CAD subjects were detected by real-time polymerase chain reaction (QRT-PCR). Associations of miR-144 expression with basic clinical characteristics and the severity of CAD were then analyzed., Results: The QRT-PCR results showed that plasma miR-144 levels were increased in CAD patients, and among CAD patients, higher SYNTAX scores and STEMI were significantly associated with higher miR-144 expression., Conclusions: Higher plasma levels of miR-144 were significantly associated with the presence as well as severity of CAD. As a potential biomarker for CAD, plasma miR-144 may be useful in predicting CAD and its severity., (© 2018 by the Association of Clinical Scientists, Inc.)