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Among the main concerns of the researchers is the comparability of the analysis methods, in order to highlight possible errors. Several statistical approaches and graphical tools are available to investigate sources of analytical error and for decision-making. In this article, we present an operative protocol for the comparison of the precision for two laboratories with the same quantitative analytical methods. This paper describes the importance of achieving sequential stages, including experimental design, familiarization with the method of optimal research analysis, quality evaluation, selection of sample, definition of acceptability criteria. Sample measurement, data analysis and evaluation, final decision and reporting are also discussed and exemplified. The reason for performing a comparison experiment is to estimate the type and magnitude of systematic error between two laboratories with the same method and to judge if the two laboratories are identical within the inherent imprecision methods or within preset analytical quality specifications. Finally, the purpose was to check if same ranges of concentrations and fluxes of pollutants are obtained using the same methods of analysis, but in two different labs.

Bis(monoacylglycero)phosphate (BMP) is a major phospholipid constituent of intralumenal membranes in late endosomes/lysosomes, where it regulates the degradation and sorting of lipid cargo. Recent observations suggest that the Batten disease-associated protein CLN5 functions as lysosomal BMP synthase. Here, we show that transacylation reactions catalyzed by cytosolic and secreted enzymes enhance BMP synthesis independently of CLN5. The transacylases identified in this study are capable of acylating the precursor lipid phosphatidylglycerol (PG), generating acyl-PG, which is subsequently hydrolyzed to BMP. Extracellularly, acyl-PG and BMP are generated by endothelial lipase in cooperation with other serum enzymes of the pancreatic lipase family. The intracellular acylation of PG is catalyzed by several members of the cytosolic phospholipase A2 group IV (PLA2G4) family. Overexpression of secreted or cytosolic transacylases was sufficient to correct BMP deficiency in HEK293 cells lacking CLN5. Collectively, our observations suggest that functionally overlapping pathways promote BMP synthesis in mammalian cells., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In mammalian cells, glycerolipids are mainly synthesized using acyl-CoA-dependent mechanisms. The acyl-CoA-independent transfer of fatty acids between lipids, designated as transacylation reaction, represents an additional mechanism for lipid remodeling and synthesis pathways. Here, we demonstrate that human and mouse phospholipase A2 group IVD (PLA2G4D) catalyzes transacylase reactions using both phospholipids and acylglycerols as substrates. In the presence of mono- and diacylglycerol (MAG and DAG), purified PLA2G4D generates DAG and triacylglycerol (TAG), respectively. The enzyme also transfers fatty acids between phospholipids and from phospholipids to acylglycerols. Overexpression of PLA2G4D in COS7 cells enhances the incorporation of polyunsaturated fatty acids into TAG stores and induces the accumulation of lysophospholipids. In the presence of exogenously added MAG, the enzyme strongly increases cellular DAG formation, while MAG levels are decreased. PLA2G4D is not or poorly detectable in commonly used cell lines. It is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines. Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, indicating that PLA2G4D is involved in the remodeling of the lipidome under inflammatory conditions. Transcriptomic analysis revealed that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling. Together, our observations demonstrate that PLA2G4D has broad substrate specificity for fatty acid donor and acceptor lipids, allowing the acyl-CoA-independent synthesis of both phospholipids and acylglycerols. Loss-of-function studies indicate that PLA2G4D affects metabolic and signaling pathways in keratinocytes, which is associated with complex lipidomic and transcriptomic alterations., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In primary hyperoxaluria type 1 excessive endogenous production of oxalate and glycolate leads to increased urinary excretion of these metabolites. Although genetic testing is the most definitive and preferred diagnostic method, quantification of these metabolites is important for the diagnosis and evaluation of potential therapeutic interventions. Current metabolite quantification methods use laborious, technically highly complex and expensive liquid, gas or ion chromatography tandem mass spectrometry, which are available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with glyoxylate generated from glycolate using recombinant mouse glycolate oxidase (GO) and glycine leads to the formation of a stable dihydroquinazoline double aromatic ring chromophore with specific peak absorption at 440 nm. The urinary limit of detection and estimated limit of quantification derived from eight standard curves were 14.3 and 28.7 µmol glycolate per mmol creatinine, respectively. High concentrations of oxalate, lactate and L-glycerate do not interfere in this assay format. The correlation coefficient between the absorption and an ion chromatography tandem mass spectrometry method is 93% with a p value < 0.00001. The Bland-Altmann plot indicates acceptable agreement between the two methods. The glycolate quantification method using conversion of glycolate via recombinant mouse GO and fusion of oABA and glycine with glyoxylate is fast, simple, robust and inexpensive. Furthermore this method might be readily implemented into routine clinical diagnostic laboratories for glycolate measurements in primary hyperoxaluria type 1., (© 2023. The Author(s).)