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The mevalonate (MVA) pathway plays a crucial role in the occurrence and progression of various diseases, such as osteoporosis, breast cancer, and lung cancer, etc. However, determining all the MVA pathway intermediates is still challenging due to their high polarity, low concentration, chelation effect with metal compartments, and poor mass spectrometric response. In this study, we established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with N 2 , N 2 , N 4 , N 4 -tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine (Tmt-PP) labeling for the simultaneous analysis of all MVA intermediates in biospecimens. Chemical derivatization significantly improved the chromatographic retention, peak shape, and detection sensitivity of the analytes. Moreover, we employed a method named mass spectrum calculation to achieve the absolute quantification of the isomers, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The established method was fully qualified and applied to explore the difference of these metabolites in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Additionally, several MVA intermediate analogs, including isopentenyl monophosphate or dimethylallyl monophosphate (IMP/DMAMP), geranyl monophosphate (GMP), 5-triphosphomevalonate (MTP), and isopentenyl triphosphate or dimethylallyl triphosphate (ITP/DMATP), were identified for the first time using a knowledge-driven prediction strategy. We further explored the tissue distribution of these novel metabolites. Overall, this work developed a sensitive quantification method for all MVA intermediates, which will enhance our understanding of the role of this pathway in various health and disease conditions. The novel metabolites we discovered warrant further investigations into their biosynthesis and biological functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The complexity of ingredients in traditional Chinese medicine (TCM) makes it challenging to clarify its efficacy in an acceptable and scientific approach. The present study was aimed to use quantification results from targeted cellular metabolomics to evaluate anti-aging efficacy of a famous Chinese medicine formula, Erzhi Wan (EZW), and screen possible effective extracts, depending on the developed strategy integrating multivariate receiver operating characteristic (ROC) curve and analytic hierarchy process (AHP). In this study, senescent NRK cells induced by D-galactose were treated with drug-containing serum of EZW and four kinds of extracts (petroleum ether, ethyl acetate, butanol and water). Intermediates of two major metabolic pathways for energy synthesis, tricarboxylic acid (TCA) cycle and glycolysis, were accurately quantified by GC-MS/MS to identify discriminate metabolites for clarifying therapeutic mechanism of EZW based on multivariate statistical analysis. Senescent and non-senescent cells were successfully distinguished using these metabolites by ROC curve analysis. Next, these metabolites were used as evaluation indexes to quantitatively reflect different effect of EZW and its extracts, according to the role of them in distinguishing groups and in conjunction with AHP. In vitro detection of senescence-associated β-galactosidase (SA-β-gal) activity was used to verify the reliability of evaluation results. The reversal after treatment of drug-containing serum of EZW and extracts was observed, and the petroleum ether extract might be the potential active extract responsible for the major anti-aging effect of EZW, which was in agreement with in vitro experiments. Altogether, metabolomics was a powerful approach for evaluation efficacy and elucidation action mechanisms of TCM. The integrated evaluation strategy in this paper with properties of high practicality, feasibility and effectivity was expected to provide a new insight into comprehensive and quantitative efficacy evaluation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because a 10 pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5 micromol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-omega-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10 pS channel. However, inhibition of CYP-omega-hydroxylation completely abolished the effect of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-omega-hydroxylation-dependent metabolites. We conclude that arachidonic acid inhibits the 10 pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-omega-hydroxylation-dependent metabolite 20-HETE.

To study the inhibition of the inwardly rectifying basolateral 50 pS potassium channels by PGE(2) we performed patch-clamp studies on the basolateral membrane of the rat kidney thick ascending limb. PGE(2)'s effect was mimicked by the selective EP1- and EP3-receptor agonist, sulprostone, but was prevented by inhibiting protein kinase-C with calphostin-C. The mitogen-activated protein kinase inhibitor PD98059 (ERK) or SB203580 (p38) increased basal channel activity; however, while neither alone prevented the inhibitory effect of PGE(2), but using both of them together completely abolished PGE(2)'s effect on channel activity. Treatment with PGE(2) stimulated phosphorylation of both p38 and ERK in primary cultures of medullary thick ascending limb cells. The PGE(2)-mediated mitogen-activated protein kinase activation was not affected by indomethacin, but was completely blocked by calphostin-C. These studies show that inhibition of basolateral 50 pS potassium channels by PGE(2) is mediated by protein kinase-C, which in turn stimulates mitogen-activated protein kinases in the thick ascending limb of the rat kidney.