Primary Hyperoxaluria Screening and Monitoring: Quantitative Measurement of Plasma Oxalate by Gas Chromatography-Mass Spectrometry With High Sensitivity.

Background: Plasma oxalate measurements can be used for the screening and therapeutic monitoring of primary hyperoxaluria. We developed a gas chromatography-mass spectrometry (GC-MS) assay for plasma oxalate measurements with high sensitivity and suitable testing volumes for pediatric populations. Methods: Plasma oxalate was extracted, derivatized, and analyzed by GC-MS. We measured the ion at m/z 261.10 to quantify oxalate and the 13C2-oxalate ion (m/z: 263.15) as the internal standard. Method validation included determination of the linear range, limit of blank, limit of detection, lower limit of quantification, precision, recovery, carryover, interference, and dilution effect. The cut-off value between primary and non-primary hyperoxaluria in a pediatric population was analyzed. Results: The detection limit was 0.78 µmol/L, and the linear range was up to 80.0 µmol/L. The between-day precision was 5.7% at 41.3 µmol/L and 13.1% at 1.6 µmol/L. The carryover was <0.2%. The recovery rate ranged from 90% to 110%. Interference analysis showed that Hb did not interfere with plasma oxalate quantification, whereas intralipids and bilirubin caused false elevation of oxalate concentrations. A cut-off of 13.9 µmol/L showed 63% specificity and 77% sensitivity, whereas a cut-off of 4.15 µmol/L showed 100% specificity and 20% sensitivity. The minimum required sample volume was 250 µL. The detected oxalate concentrations showed interference from instrument conditioning, sample preparation procedures, medications, and various clinical conditions. Conclusions: GC-MS is a sensitive assay for quantifying plasma oxalate and is suitable for pediatric patients. Plasma oxalate concentrations should be interpreted in a clinical context.

A highly accurate mass spectrometry method for the quantification of phenylalanine and tyrosine on dried blood spots: Combination of liquid chromatography, phenylalanine/tyrosine-free blood calibrators and multi-point/dynamic calibration.

BACKGROUND: Measurement of quantitative levels of phenylalanine and tyrosine in blood is an essential test for the diagnosis of and monitoring genetic disorders associated with phenylalanine metabolism, such as phenylketonuria (PKU), tyrosinemia, and defects of tetrahydrobiopterin synthesis and recycling. We developed a highly accurate and fast liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantification of phenylalanine and tyrosine on dried blood spot (DBS). We also designed a performance score system to evaluate various calibration methods in matrix matched material. METHODS: Phenylalanine/tyrosine-free whole blood was used to make accurate and stable DBS calibrators. Six calibrators cover the range of 0-1000 µmol/L. Underivatized phenylalanine and tyrosine were extracted and measured by LC-MS/MS. Precision, accuracy, limit of quantification, recovery and carryover were validated. External quality assurance materials were also used to evaluate performance of multi-point calibrations and single-point calibrations. RESULTS: The run time was 4.5-minute. Accuracy analysis showed good agreement with reference materials. Precision, recovery, and the lower and upper limit of quantification met the criteria. When phenylalanine and tyrosine concentrations were less than 150 µmol/L, the 5-point calibration without the calibrator of 1000 µmol/L had the best performance. When the concentrations were > 250 µmol/L, the single-point calibration of 500 µmol/L had the best performance. CONCLUSION: We developed a simple, fast and highly accurate method for the detection of phenylalanine and tyrosine on DBS, with chromatographic separation and underivatized analysis. Based on the calibration performance, a 6-point calibration method is satisfying for this test. An optional dynamic calibration method, which includes 6-point calibration, 5-point calibration and single-point calibration, can further increase test reliability.

Non-alcoholic fatty liver disease in mice with heterozygous mutation in TMED2.

The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. Members of the TMED family serve as cargo receptors implicated in selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Deletion or mutations of Tmed genes in yeast and Drosophila results in ER-stress and activation of the unfolded protein response (UPR). The UPR leads to expression of genes and proteins important for expanding the folding capacity of the ER, degrading misfolded proteins, and reducing the load of new proteins entering the ER. The UPR is activated in non-alcoholic fatty liver disease (NAFLD) in human and mouse and may contribute to the development and the progression of NAFLD. Tmed2, the sole member of the vertebrate Tmed ß subfamily, exhibits tissue and temporal specific patterns of expression in embryos and developing placenta but is ubiquitously expressed in all adult organs. We previously identified a single point mutation, the 99J mutation, in the signal sequence of Tmed2 in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Histological and molecular analysis of livers from heterozygous mice carrying the 99J mutation, Tmed299J/+, revealed a requirement for TMED2 in liver health. We show that Tmed299J/+ mice had decreased levels of TMED2 and TMED10, dilated endoplasmic reticulum membrane, and increased phosphorylation of eIF2α, indicating ER-stress and activation of the UPR. Increased expression of Srebp1a and 2 at the newborn stage and increased incidence of NAFLD were also found in Tmed299J/+ mice. Our data establishes Tmed299J/+ mice as a novel mouse model for NAFLD and supports a role for TMED2 in liver health.

Design, synthesis and biological evaluation of Lenalidomide derivatives as tumor angiogenesis inhibitor.

Lenalidomide is a type of immunomodulatory agent with anti-tumor activity by mainly expressed in the anti-angiogenesis. In order to enhance the pharmacological activity of Lenalidomide, a series of Lenalidomide derivatives were designed as tumor angiogenesis inhibitors. The potential anti-angiogenesis targets of Lenalidomide derivatives were virtual screened on Auto-Dock 4.0 by using reverse docking method. The six target proteins, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, BCR-ABL tyrosine kinase, p38 mitogen activated protein kinase and metal protein kinase, were chosen as the targets. The Lenalidomide derivatives were synthesized by alkylated, acylated or sulfonylated Lenalidomide and verified by the 1H NMR, 13C NMR and LC-MS. Their anti-cancer activities were detected by using CCK-8 in the esophageal carcinoma cell line EC9706. The results indicate that the inhibitory activities of Lenalidomide derivatives were higher than that of Lenalidomide.

Quantitative Analysis of Total Plasma Homocysteine by LC-MS/MS.

Homocysteine is a nonessential, sulfur-containing amino acid involved in one-carbon (folate) metabolism. A number of inherited and acquired conditions cause increased accumulation of this metabolite in blood (homocysteinemia) and other biofluids. Homocysteinemia is a risk factor for cardiovascular disease, including recurrent thrombosis. Accurate measurement of total plasma homocysteine is an important element in the diagnostic evaluation of these disorders. While a number of different methods have been developed for this purpose, the focus of this unit will be on a specific technique utilizing liquid chromatography-tandem mass spectrometry, which provides several advantages in terms of speed, sensitivity, and specificity.

Diethyl (E)-2,3-bis-[(E)-(2-methyl-2-phenyl-hydrazin-1-yl-idene)meth-yl]but-2-enedioate.

The complete mol-ecule of the title compound, C24H28N4O4, is generated by crystallographic inversion symmetry. The ethyl side chain is disordered over two sets of sites in a 0.57 (4):0.43 (4) ratio. The dihedral angles between the methyl-idene group and the phenyl ring and ester side chain (major conformation) are 7.61 (8) and 86.95 (8)°, respectively. In the crystal, mol-ecules are linked via C-H⋯O hydrogen bonds, forming corrugated sheets lying parallel to (010).

Prosaposin sorting is mediated by oligomerization.

The compartmental nature of eukaryotic cells requires sophisticated mechanisms of protein sorting. Prosaposin, the precursor of four sphingolipid activator proteins, is transported from the trans-Golgi network (TGN) to lysosomes as a partially glycosylated (65 kDa) protein with high-mannose/hybrid oligosaccharides. Prosaposin is also found in the extracellular space where it is secreted as a fully glycosylated (70 kDa) protein composed of complex glycans. Although the trafficking of prosaposin to lysosomes is known to be mediated by sortilin, the mechanism of secretion of this protein is still unknown. In this study, we report that prosaposin may covalently aggregate into oligomers. Our results demonstrate that while prosaposin oligomers are secreted into the extracellular space, monomeric prosaposin remains inside the cell bound to sortilin. We also found that deletion of the C-terminus of prosaposin, previously shown to block its lysosomal transport, did not abolish its oligomerization and secretion. On the other hand, elimination of the N-terminus and of each saposin domain inhibited its oligomerization and resulted in its retention as a fully glycosylated protein. In conclusion, we are reporting for the first time that oligomerization of prosaposin is crucial for its entry into the secretory pathway.

A stretch of 17 amino acids in the prosaposin C terminus is critical for its binding to sortilin and targeting to lysosomes.

Prosaposin, the precursor of four lysosomal cofactors required for the hydrolysis of sphingolipids, is transported to the lysosomes via the alternative receptor, sortilin. In this study, we identified a specific domain of 17 amino acids within the C terminus of prosaposin involved in binding to this sorting receptor. We generated six prosaposin deletion constructs and examined the effect of truncation by coimmunoprecipitation and confocal microscopy. The experiments revealed that the first half of the prosaposin C terminus (aa 524-540), containing a saposin-like motif, was required and necessary to bind sortilin and to transport it to the lysosomes. Based on this result, we introduced twelve site-directed point mutations within the first half of the C terminus. Although the interaction of prosaposin with sortilin was pH dependent, the mutation of hydrophilic amino acids that usually modulate pH-dependent protein interactions did not affect the binding of prosaposin to sortilin. Conversely, a tryptophan (W530) and two cysteines (C528 and C536) were essential for its interaction with sortilin and for its transport to the lysosomes. In conclusion, our investigation demonstrates that a saposin-like motif within the first half of the prosaposin C terminus contains the sortilin recognition site.

Sortilin and prosaposin localize to detergent-resistant membrane microdomains.

Most soluble lysosomal hydrolases are sorted in the trans-Golgi network (TGN) and delivered to the lysosomes by the mannose 6-phosphate receptor (M6PR). However, the non-enzymic sphingolipid activator protein (SAP), prosaposin, as well as certain soluble lysosomal hydrolases, is sorted and trafficked to the lysosomes by sortilin. Based on previous results demonstrating that prosaposin requires sphingomyelin to be targeted to the lysosomes, we hypothesized that sortilin and its ligands are found in detergent-resistant membranes (DRMs). To test this hypothesis we have analyzed DRM fractions and demonstrated the presence of sortilin and its ligand, prosaposin. Our results showed that both the M6PR and its cargo, cathepsin B, were also present in DRMs. Cathepsin H has previously been demonstrated to interact with sortilin, while cathepsin D interacts with both sortilin and the M6PR. Both of these soluble lysosomal proteins were also found in DRM fractions. Using sortilin shRNA we have showed that prosaposin is localized to DRM fractions only in the presence of sortilin. These observations suggest that in addition to interacting with the same adaptor proteins, such as GGAs, AP-1 and retromer, both sortilin and the M6PR localize to similar membrane platforms, and that prosaposin must interact with sortilin to be recruited to DRMs.