Association between body mass index and electrocardiogram indices: A Mendelian randomization study.

INTRODUCTION: Cardiovascular diseases (CVDs) remain a global health concern, and body mass index (BMI) is known to be associated with an increased risk of CVD, but the exact mechanisms underlying this relationship remain unclear. This study employs Mendelian randomization (MR) to investigate the causal association between BMI and electrocardiogram (ECG) indices, providing insights into potential pathways linking obesity to CVD. METHODS: We conducted a comprehensive MR study utilizing large-scale genetic and ECG data from diverse populations. Instrumental variables were selected from genome-wide association studies, ensuring their relevance to BMI. Causal relationships between BMI and ECG indices, including P wave duration, PR interval, QRS duration, and QT interval, were assessed using various MR methods, with inverse variance weighted (IVW) considered as the primary analysis. RESULTS: Our MR analysis revealed a significant positive causal association between higher BMI and P wave duration (ß = 8.078, 95% CI: 5.322 to 10.833, p < 0.001), suggesting a potential mechanism through which higher BMI may contribute to arrhythmogenic risks. However, no significant causal associations were observed between BMI and PR interval, QRS duration, or QT interval (all p > 0.005). In addition, our study also found that there is no horizontal pleiotropy between BMI and P wave duration, PR interval, QRS duration, and QT interval, suggesting that the conclusions of this study are robust. CONCLUSION: This study supports a causal relationship between elevated BMI and prolonged P wave duration, a marker of increased atrial arrhythmogenic risk. Further investigations are still needed to elucidate the underlying mechanisms.

PU.1 promotes development of rheumatoid arthritis via repressing FLT3 in macrophages and fibroblast-like synoviocytes.

OBJECTIVES: To uncover the function and underlying mechanism of an essential transcriptional factor, PU.1, in the development of rheumatoid arthritis (RA). METHODS: The expression and localisation of PU.1 and its potential target, FMS-like tyrosine kinase 3 (FLT3), in the synovium of patients with RA were determined by western blot and immunohistochemical (IHC) staining. UREΔ (with PU.1 knockdown) and FLT3-ITD (with FLT3 activation) mice were used to establish collagen antibody-induced arthritis (CAIA). For the in vitro study, the effects of PU.1 and FLT3 on primary macrophages and fibroblast-like synoviocytes (FLS) were investigated using siRNAs. Mechanistically, luciferase reporter assays, western blotting, FACS and IHC were conducted to show the direct regulation of PU.1 on the transcription of FLT3 in macrophages and FLS. Finally, a small molecular inhibitor of PU.1, DB2313, was used to further illustrate the therapeutic effects of DB2313 on arthritis using two in vivo models, CAIA and collagen-induced arthritis (CIA). RESULTS: The expression of PU.1 was induced in the synovium of patients with RA when compared with that in osteoarthritis patients and normal controls. FLT3 and p-FLT3 showed opposite expression patterns compared with PU.1 in RA. The CAIA model showed that PU.1 was an activator, whereas FLT3 was a repressor, of the development of arthritis in vivo. Moreover, results from in vitro assays were consistent with the in vivo results: PU.1 promoted hyperactivation and inflammatory status of macrophages and FLS, whereas FLT3 had the opposite effects. In addition, PU.1 inhibited the transcription of FLT3 by directly binding to its promoter region. The PU.1 inhibitor DB2313 clearly alleviated the effects on arthritis development in the CAIA and CIA models. CONCLUSIONS: These results support the role of PU.1 in RA and may have therapeutic implications by directly repressing FLT3. Therefore, targeting PU.1 might be a potential therapeutic approach for RA.

The application of HER2 and CD47 CAR-macrophage in ovarian cancer.

BACKGROUND: The chimeric antigen receptor (CAR)-T therapy has a limited therapeutic effect on solid tumors owing to the limited CAR-T cell infiltration into solid tumors and the inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Macrophage is an important component of the innate and adaptive immunity, and its unique phagocytic function has been explored to construct CAR macrophages (CAR-Ms) against solid tumors. This study aimed to investigate the therapeutic application of CAR-Ms in ovarian cancer. METHODS: In this study, we constructed novel CAR structures, which consisted of humanized anti-HER2 or CD47 scFv, CD8 hinge region and transmembrane domains, as well as the 4-1BB and CD3ζ intracellular domains. We examined the phagocytosis of HER2 CAR-M and CD47 CAR-M on ovarian cancer cells and the promotion of adaptive immunity. Two syngeneic tumor models were used to estimate the in vivo antitumor activity of HER2 CAR-M and CD47 CAR-M. RESULTS: We constructed CAR-Ms targeting HER2 and CD47 and verified their phagocytic ability to ovarian cancer cells in vivo and in vitro. The constructed CAR-Ms showed antigen-specific phagocytosis of ovarian cancer cells in vitro and could activate CD8+ cytotoxic T lymphocyte (CTL) to secrete various anti-tumor factors. For the in vivo model, mice with human-like immune systems were used. We found that CAR-Ms enhanced CD8+ T cell activation, affected tumor-associated macrophage (TAM) phenotype, and led to tumor regression. CONCLUSIONS: We demonstrated the inhibition effect of our constructed novel HER2 CAR-M and CD47 CAR-M on target antigen-positive ovarian cancer in vitro and in vivo, and preliminarily verified that this inhibitory effect is due to phagocytosis, promotion of adaptive immunity and effect on tumor microenvironment.

High-security constellation shaped self-homodyne coherent system with 4-D joint encryption.

In recent years, the self-homodyne coherent (SHC) system and the constellation shaping (CS) technique have drawn considerable attention due to their abilities to further improve the transmission capacity for various scenarios. From the security point of view, the CS technique and the SHC infrastructure also provide new dimensions for encryption. We propose a high-security and reliable SHC system based on the CS technique and the digital chaos. With a four-dimensional hyperchaotic system, chaotic sequences are generated and used for the exclusive or operation, chaotic constant composition distribution matching, phase disturbance, and optical-layer time-delay disturbance. Moreover, 64-ary circular quadrature amplitude modulation (64CQAM) format is adopted for transmission due to its advantages of sensitivity to phase noise, immunity to conventional digital signal processing, and ability of time-mismatch masking, which is verified by simulation in a SHC system. Last, we conduct an experimental verification in a 20GBaud probabilistically shaped 64CQAM SHC system. Consequently, with a large-linewidth laser source, optical-layer security can be protected by time mismatches of tens of picoseconds. And the digital-layer security is protected by an enormous key space of 10127. The proposed scheme can provide reliable real-time encryption for the optical fiber transmission, serving as a potential candidate for the future high-capacity inter/intra-datacenter security interconnect.

Blind SOP recovery of eigenvalue communication system based on a nonlinear Fourier transform.

Owing to the random birefringence of optical fibers, the recovery of the state of polarization (SOP) is urgently needed, especially in the nonlinear spectrum division multiplexing transmissions. Based on the variance of the polarization power ratio among symbols as the cost function, we propose a novel algorithm for the blind SOP recovery of eigenvalue communications. In the single eigenvalue transmissions with phase-shift keying or 16-ary amplitude and phase-shift keying constellations, at least 25.3 dB polarization extinction ratio can be achieved by using a block length of 30, even under 7 dB OSNR condition. It also shows that the proposed algorithm can be employed in multi-eigenvalue NFDM transmissions and full-spectrum modulated NFDM system. In the experiment, our proposed algorithm performs the same as the training symbol based method in back-to-back and less than 3000 km fiber link conditions; a maximum performance gain of 1.6 dB was obtained in ultra-long-haul condition (4300 km). It also shows that the impact of the polarization mode dispersion of a single-mode fiber on the algorithm is negligible.

Enabling cost-effective high-performance vibration sensing in digital subcarrier multiplexing systems.

Enabling communication networks with sensing functionality has attracted significant interest lately. The digital subcarrier multiplexing (DSCM) technology is widely promoted in short-reach scenarios for its inherent flexibility of fine-tuning the spectrum. Its compatibility with large-scale as-deployed coherent architectures makes it particularly suited for cost-sensitive integrated sensing and communication applications. In this paper, we propose a scheme of spectrally integrating the digital linear frequency modulated sensing signal into DSCM signals to achieve simultaneous sensing and communication through shared transmitter. Consequently, this cost-effective scheme has been demonstrated to achieve 100-Gb/s dual-polarization quadrature phase-shift keying (DP-QPSK) and 200-Gb/s dual-polarization 16-ary quadrature amplitude modulation (DP-16QAM) transmission with a distributed acoustic sensing sensitivity of 69 pε/Hz and 88 pε/Hz respectively, at a spatial resolution of 4 m.

PDM-SHD NFDM transmission with envelope-detection feedback polarization tracking and optical injection locking.

Nonlinear frequency division multiplexing (NFDM) systems, especially the eigenvalue communications have the potential to overcome the nonlinear Shannon capacity limit. However, the baud rate of eigenvalue communications is typically restricted to a few GBaud, making it challenging to mitigate laser frequency impairments such as the phase noise and frequency offset (FO) using digital signal processing (DSP) algorithms in intradyne detections (IDs). Therefore, we introduce the polarization division multiplexing-self-homodyne detection (PDM-SHD) into the NFDM link, which could overcome the impact of phase noise and FO by transmitting a pilot carrier originating from the transmitter laser to the receiver through the orthogonal polarization state of signal. To separate the signal from the carrier at the receiver, a carrier to signal power ratio (CSPR) unrestricted adaptive polarization controlling strategy is proposed and implemented by exploiting the optical intensity fluctuation of the light in a particular polarization rather than its direct optical power as the feedback. Optical injection locking (OIL) is used subsequently to amplify optical power of pilot carrier and mitigate the impact of signal-signal beat interference (SSBI). Additionally, the effects of cross-polarization modulation (XPolM) and modulation instability (MI) in long haul transmission are explored and inhibited. The results show that the tolerable FO range is about 3.5 GHz, which is 17 times larger than the ID one. When 16-amplitude phase shift keying (APSK) or 64-APSK constellations are used, identical Q-factor performance can be obtained by using distributed feedback (DFB, ∼10 MHz) laser, external cavity laser (ECL, ∼100kHz), or fiber laser (FL, ∼100 Hz), respectively, which demonstrates that our proposed PDM-SHD eigenvalue communication structure is insensitive to the laser linewidth. Under the impact of cycle slip, the Q-factor difference of 16-APSK signal between the ECL-ID system and ECL-SHD system can be up to 8.73 dB after 1500 km transmission.

Endogenous BOTDA in the self-homodyne coherent detection transmission system for communication-integrated datacenter temperature monitoring.

This Letter demonstrates the high compatibility of the self-homodyne coherent detection (SHCD) transmission system with the Brillouin optical time-domain analyzer (BOTDA). By fully utilizing the remote delivered local oscillator (LO) light of the transmission system, the first, to the best of our knowledge, endogenously integrated BOTDA subsystem is achieved. The remote delivery of the homologous laser source in the SHCD system ensures the frequency match between the probe light and the pump light of the BOTDA. Furthermore, an injection-locked distributed feedback (DFB) laser is employed to amplify the LO and eliminate the impact induced by the Brillouin gain. The experiment demonstrates that a 16-km distributed temperature sensing based on BOTDA can be insensibly emerged into a 50-Gbaud DP-16QAM SHCD transmission system (400 Gbps/λ/core), achieving a spatial resolution of 3 meters and a temperature accuracy of 1°C. Remarkably, the auxiliary sensing module has negligible impact on the transmission.

Inter-annual and inter-species tree growth explained by phenology of xylogenesis.

Wood formation determines major long-term carbon (C) accumulation in trees and therefore provides a crucial ecosystem service in mitigating climate change. Nevertheless, we lack understanding of how species with contrasting wood anatomical types differ with respect to phenology and environmental controls on wood formation. In this study, we investigated the seasonality and rates of radial growth and their relationships with climatic factors, and the seasonal variations of stem nonstructural carbohydrates (NSC) in three species with contrasting wood anatomical types (red oak: ring-porous; red maple: diffuse-porous; white pine: coniferous) in a temperate mixed forest during 2017-2019. We found that the high ring width variability observed in both red oak and red maple was caused more by changes in growth duration than growth rate. Seasonal radial growth patterns did not vary following transient environmental factors for all three species. Both angiosperm species showed higher concentrations and lower inter-annual fluctuations of NSC than the coniferous species. Inter-annual variability of ring width varied by species with contrasting wood anatomical types. Due to the high dependence of annual ring width on growth duration, our study highlights the critical importance of xylem formation phenology for understanding and modelling the dynamics of wood formation.

Insights into source/sink controls on wood formation and photosynthesis from a stem chilling experiment in mature red maple.

Whether sources or sinks control wood growth remains debated with a paucity of evidence from mature trees in natural settings. Here, we altered carbon supply rate in stems of mature red maples (Acer rubrum) within the growing season by restricting phloem transport using stem chilling; thereby increasing carbon supply above and decreasing carbon supply below the restrictions, respectively. Chilling successfully altered nonstructural carbon (NSC) concentrations in the phloem without detectable repercussions on bulk NSC in stems and roots. Ring width responded strongly to local variations in carbon supply with up to seven-fold differences along the stem of chilled trees; however, concurrent changes in the structural carbon were inconclusive at high carbon supply due to large local variability of wood growth. Above chilling-induced bottlenecks, we also observed higher leaf NSC concentrations, reduced photosynthetic capacity, and earlier leaf coloration and fall. Our results indicate that the cambial sink is affected by carbon supply, but within-tree feedbacks can downregulate source activity, when carbon supply exceeds demand. Such feedbacks have only been hypothesized in mature trees. Consequently, these findings constitute an important advance in understanding source-sink dynamics, suggesting that mature red maples operate close to both source- and sink-limitation in the early growing season.

Unequally spaced PAM-4 signaling enabled sensitivity enhancement of a simplified coherent receiver applied in a UDWDM-PON.

We experimentally demonstrate a 4 × 10 Gb/s cost-effective coherent ultra-dense wavelength division multiplexing passive optical network (UDWDM-PON) by the use of unequally-spaced 4-level pulse-amplitude modulation (UES-PAM-4) signaling. Because of the advantages of simple architecture and low cost, the simplified coherent receiver (SCR) based on the transmitted signal diversity (TS-D) has been reported, but its receiver sensitivity is constrained by the severe noise arising in the higher level of conventional PAM-4 signals. Here, we first experimentally demonstrate the UES-PAM-4 signaling for the SCR based on the TS-D, by altering the PAM-4 level spacing and the decision threshold through the gradient descent algorithm (GDA). Consequently, we can experimentally achieve -30.1 dBm RS for single wavelength at the bit-error ratio (BER) of 3.8 × 10-3. Compared with the conventional equally-spaced PAM-4 (ES-PAM-4) signaling, 1.3 dB RS enhancement can be secured after the 20-km standard single-mode fiber (SSMF) transmission. Meanwhile, the UES-PAM-4 signaling is experimentally verified for 4 × 10 Gb/s UDWDM-PON. An average RS of -29.6 dBm and 32.6 dB power budget are obtained after the 20-km SSMF transmission. The proposed UES-PAM-4 signaling with the RS enhancement is a promising candidate for the UDWDM-PON by utilizing the existing optical distribution network (ODN).

Modeling and mitigation of polarization crosstalk-induced nonlinearity for the polarization-multiplexed carrier self-homodyne system.

In this Letter, we analytically model the impact of polarization crosstalk in the polarization-multiplexed carrier self-homodyne (PMC-SH) system with adaptive polarization control technology. When the optical paths of the signal and local oscillator (LO) are matched well, it is found that the polarization crosstalk results in a nonlinear shift on the constellation. Thus, we further propose a compensation scheme based on a low-complexity polynomial nonlinear equalizer (PNLE). Both simulation and experimental results validate our theoretical analysis. Moreover, the proposed PNLE-based compensation scheme achieves up to 1.23 dB tolerance improvement with respect to polarization crosstalk for 20 Gbaud 64QAM in the experiment.

Puerarin attenuates intracerebral hemorrhage-induced early brain injury possibly by PI3K/Akt signal activation-mediated suppression of NF-κB pathway.

Intracerebral hemorrhage (ICH) can induce intensively oxidative stress, neuroinflammation, and brain cell apoptosis. However, currently, there is no highly effective treatment available. Puerarin (PUE) possesses excellent neuroprotective effects by suppressing the NF-κB pathway and activating the PI3K/Akt signal, but its role and related mechanisms in ICH-induced early brain injury (EBI) remain unclear. In this study, we intended to observe the effects of PUE and molecular mechanisms on ICH-induced EBI. ICH was induced in rats by collagenase IV injection. PUE was intraperitoneally administrated alone or with simultaneously intracerebroventricular injection of LY294002 (a specific inhibitor of the PI3K/Akt signal). Neurological deficiency, histological impairment, brain edema, hematoma volume, blood-brain barrier destruction, and brain cell apoptosis were evaluated. Western blot, immunohistochemistry staining, reactive oxygen species (ROS) measurement, and enzyme-linked immunosorbent assay were performed. PUE administration at 50 mg/kg and 100 mg/kg could significantly reduce ICH-induced neurological deficits and EBI. Moreover, PUE could notably restrain ICH-induced upregulation of the NF-κB pathway, pro-inflammatory cytokines, ROS level, and apoptotic pathway and activate the PI3K/Akt signal. However, LY294002 delivery could efficaciously weaken these neuroprotective effects of PUE. Overall, PUE could attenuate ICH-induced behavioral defects and EBI possibly by PI3K/Akt signal stimulation-mediated inhibition of the NF-κB pathway, and this made PUE a potential candidate as a promising therapeutic option for ICH-induced EBI.

Manipulating phloem transport affects wood formation but not local nonstructural carbon reserves in an evergreen conifer.

How variations in carbon supply affect wood formation remains poorly understood in particular in mature forest trees. To elucidate how carbon supply affects carbon allocation and wood formation, we attempted to manipulate carbon supply to the cambial region by phloem girdling and compression during the mid- and late-growing season and measured effects on structural development, CO2 efflux and nonstructural carbon reserves in stems of mature white pines. Wood formation and stem CO2 efflux varied with a location relative to treatment (i.e., above or below the restriction). We observed up to twice as many tracheids formed above versus below the treatment after the phloem transport manipulation, whereas the cell-wall area decreased only slightly below the treatments, and cell size did not change relative to the control. Nonstructural carbon reserves in the xylem, needles and roots were largely unaffected by the treatments. Our results suggest that low and high carbon supply affects wood formation, primarily through a strong effect on cell proliferation, and respiration, but local nonstructural carbon concentrations appear to be maintained homeostatically. This contrasts with reports of decoupling of source activity and wood formation at the whole-tree or ecosystem level, highlighting the need to better understand organ-specific responses, within-tree feedbacks, as well as phenological and ontogenetic effects on sink-source dynamics.

Digital in-service relative time delay estimation for SDM self-homodyne coherent systems.

For space division multiplexing self-homodyne coherent systems, we propose a novel digital in-service relative time delay (RTD) estimation method without any additional optoelectronic device. Taking advantage of the frequency-domain periodicity of the colored frequency modulation noise, we manage to find the peak with location reflecting the RTD in its autocorrelation function (ACF). The peak to average ratio is further enhanced by leveraging a low-pass differential finite impulse response filter for robust identification. By simulations, the method is validated to be feasible for various linewidths, formats (16QAM, 32QAM and 64QAM), and links up to 80 km. Particularly, it is proved to be inherently compatible with large-linewidth low-cost lasers for the 10-km link. Also, for a low-complexity implementation, we discuss the way to reduce the number of points used to calculate the ACF while maintaining the same dynamic range. Furthermore, we demonstrate a 50-GBaud 16-QAM experiment to investigate its performances. With received optical power varying from -11 dBm to -17 dBm, 216 points are sufficient to provide an estimation accuracy of standard deviation (STD) less than 0.089 ns for the RTD range of [2.6, 491.0 ns]. The STD can be lowered to 0.036 ns by adopting 218 points. Especially, at -11-dBm ROP, the highest performance has been achieved with an accuracy smaller than the symbol period (0.018-ns STD) and a RTD range of [1.5, 491.0 ns].

Direct response of tree growth to soil water and its implications for terrestrial carbon cycle modelling.

Wood growth constitutes the main process for long-term atmospheric carbon sequestration in vegetation. However, our understanding of the process of wood growth and its response to environmental drivers is limited. Current dynamic global vegetation models (DGVMs) are mainly photosynthesis-driven and thus do not explicitly include a direct environmental effect on tree growth. However, physiological evidence suggests that, to realistically model vegetation carbon allocation under increased climatic stressors, it is crucial to treat growth responses independently from photosynthesis. A plausible growth response function suitable for global simulations in DGVMs has been lacking. Here, we present the first soil water-growth response function and parameter range for deciduous and evergreen conifers. The response curve was calibrated against European larch and Norway spruce in a dry temperate forest in the Swiss Alps. We present a new data-driven approach based on a combination of tree ring width (TRW) records, growing season length and simulated subdaily soil hydrology to parameterize ring width increment simulations. We found that a simple linear response function, with an intercept at zero moisture stress, used in growth simulations reproduced 62.3% and 59.4% of observed TRW variability for larch and spruce respectively and, importantly, the response function slope was much steeper than literature values for soil moisture effects on photosynthesis and stomatal conductance. Specifically, we found stem growth stops at soil moisture potentials of -0.47 MPa for larch and -0.66 MPa for spruce, whereas photosynthesis in trees continues down to -1.2 MPa or lower, depending on species and measurement method. These results are strong evidence that the response functions of source and sink processes are indeed very different in trees, and need to be considered separately to correctly assess vegetation responses to environmental change. The results provide a parameterization for the explicit representation of growth responses to soil water in vegetation models.

Geometric shaping optimization of 64-APSK constellation in discrete nonlinear frequency division multiplexing systems.

We experimentally demonstrated a geometric shaped (GS) 64-ary amplitude phase shift keying (64-APSK) eigenvalue transmission. The signal is modulated on the scatter coefficient of a single eigenvalue and linear minimum mean square error (LMMSE) estimator is used to reduce the noise. The channel response is estimated by transmitting a normally distributed 64-APSK constellation through a communication link. Based on the polar coordinates distribution of the received constellation, the diameter distributions for each circle can be obtained so that circles with larger noise can obtain larger judgment width. After optimization, the experimental results show that the Q-factor gain is 1.13 dB under 22 dB received optical signal to noise ratio (OSNR) configuration and 0.88 dB after 900 km transmission compared with normally distributed APSK configuration.

All-optical polarization split of the signal and LO for a bi-directional self-homodyne coherent system.

In this Letter, we propose a cost-efficient bi-directional (BiDi) polarization-multiplexed self-homodyne coherent detection (SHCD) system, in which only one fiber link and one adaptive polarization controller (APC) are required. By employing the correlation of the state of polarization (SOP) between the upstream and downstream light, one APC is capable of stabilizing SOPs of the counterpropagating waves at the same time. The signal and local oscillator (LO) can be optically split by a polarization beam splitter (PBS), relaxing pressure of the digital signal processing (DSP) and simplifying the coherent receiver. The impact induced by polarization cross talk and delay mismatch is collectively investigated by theoretical analysis and simulation. Finally, the proposed scheme is experimentally verified through 120 Gbit/s 16-quadrature amplitude modulation (16-QAM) transmission, achieving a satisfying laser-linewidth tolerance of 10 MHz and a polarization rotation tolerance of up to 45 rad/s.

Measurement of urine albumin by liquid chromatography-isotope dilution tandem mass spectrometry and its application to value assignment of external quality assessment samples and certification of reference materials.

OBJECTIVES: Urine albumin is measured in clinical laboratories by immunoturbidimetry. However, large biases are observed among the different routine methods. To standardize the measurement of urine albumin, a reference measurement procedure (RMP) and urine albumin certified reference materials (CRMs) are needed. METHODS: A candidate RMP for urine albumin based on liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) using human serum albumin as calibration standard was developed. Isotope-labeled human albumin was used as internal standard. Urine samples were digested using trypsin and eight resulting "signature" peptides of albumin were quantified by LC-IDMS/MS. The candidate RMP was employed in value assignment of external quality assessment (EQA) samples and certification of urine albumin reference materials. The commutability of the developed CRMs was assessed against patient samples. RESULTS: The candidate RMP (recovery 101.5-103.2% and CV 1.2-3.3% at about 7-40 mg/L) met optimal performance goal. The lower limit of quantification was 0.03 mg/L as determined by signal-to-noise method. The EQA results from clinical laboratories using different immunoturbidimetric methods were generally comparable with assigned target values determined by the candidate RMP, with albumin concentrations ranging from 5 to 226 mg/L. Urine albumin reference materials (two levels) certified using the candidate RMP showed good commutability in a preliminary study. CONCLUSIONS: With optimal method precision and trueness, as well as comparability with routine methods, the developed RMP may be used for value assignment of EQA samples or certification of reference materials, which are important pillars in urine albumin method standardization.

Determination of purity values of amino acid reference materials by mass balance method: an approach to the quantification of related structure impurities.

A systematic procedure for the determination of purity values of amino acid reference materials was developed by use of mass balance method where four categories of impurities (related structure impurities (RSIs), water, organic solvent residue (OSR), and non-volatile residue (NVR)) were quantified separately. The amount of RSIs was determined using a combination of three quantification methods. To ensure metrological traceability in the determination of RSIs, at least one such impurity in each candidate amino acid reference material was quantified using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS). Other RSIs were determined using external calibration liquid chromatography-tandem mass spectrometry (LC-MS/MS) or o-phthaldialdehyde (OPA) derivatization, followed by liquid chromatography-ultraviolet (LC-UV) measurement. As the UV absorption of most RSIs came basically from the same chromophore after OPA derivatization, a relative peak area approach was used in the LC-UV method to quantify the amount of RSIs by comparing their peak areas with that of a reference RSI. The reference RSI was pre-selected and the amount determined by LC-IDMS/MS separately. The absence of D-amino acids was confirmed using Marfey's reagent derivatization, followed by LC-UV analysis. The amounts of water, OSR, and NVR were measured using Karl Fischer coulometry, gas chromatography-mass spectrometry (GC-MS) and thermogravimetry, respectively. By using this procedure, four amino acid (L-valine, L-leucine, L-isoleucine, and L-phenylalanine) certified reference materials (CRMs) were developed from the candidate materials. The homogeneity and stability of the CRMs were demonstrated by use of LC-IDMS/MS or OPA-LC-UV method, following the principles in ISO 17034 and ISO Guide 35.Graphical abstract.