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A new method for the determination of free calcium concentration in human plasma was developed by online coupling capillary electrophoresis (CE) with inductively coupled plasma optical emission spectrometry (ICP-OES). Baseline separation of calcium-containing species was achieved by CE-ICP-OES in a 120-cm-long capillary with 100-[micro]m internal diameter, at 20 kV applied voltage, with a 30 mmol/L Tris-HCl buffer at pH 7.4. A total of eight calcium-containing species were found in human plasma; the concentration of free calcium ion was found to be 41.9 mg/L. The concentrations of calcium for other seven calcium species, estimated from the calibration against [Ca.sup.2[+ or -] ] standard, were 3.14-15.6 mg/L. The precision (RSD, n = 10) ranged from 1.2 to 2.7% for the migration time and 2.8 to 3.9% for the peak area. The developed method was also applied to analyze plasma samples with recovery ranged from 94.5 to 102% for samples spiked with 40 mg/L free [Ca.sup.2[+ or -] ] ion.

A rapid and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous determination of thirteen bioactive components (gallic acid, protocatechuic acid, puerarin, p‐hydroxycinnamic acid, daidzin, ononin, daidzein, naringenin, genistein, apigenin, formononetin, biochanin A, and β‐sitosterol) of Radix Puerariae extract in rat plasma and tissues. The plasma and tissues samples were pretreated by protein precipitation extraction, and umbelliferone and rutin were used as internal standards. Sample separation was performed on a ZORBAX RRHD Eclipse plus C18 column (2.1 mm × 50 mm, 1.8 µm, Agilent) with a mobile phase consisting of methanol–water (containing 0.1% formic acid). The mass spectrometry analysis was conducted in positive and negative ionization modes with multiple reaction monitoring. The lower limit of quantitation range for the 13 analytes was 0.2−35 ng/mL. The intra‐ and inter‐day precision of all the analytes were less than 10.92%, with an accuracy ranging from −13.10 to 11.96%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied to pharmacokinetic and tissue distribution study of the 13 bioactive components in rats after oral administration of R. Puerariae extract. [ABSTRACT FROM AUTHOR]

S-adenosylhomocysteine ( SAH), formed after donation of the methyl group of S-adenosylmethionine ( SAM) to a methyl acceptor, is reversibly hydrolyzed to adenosine ( ADO) and homocysteine ( HCY) by S-adenosylhomocysteine hydrolase ( SAHH). In chestnut blight fungus ( Cryphonectria parasitica), sahh, a hypovirus-regulated gene that encodes a deduced SAHH protein was shown to have an SAHH enzymatic activity in vitro. Deletion of sahh resulted in the increased accumulation of intracellular SAH and SAM but decreased ADO, and a remarkably increased accumulation of transcripts that encode adenosine kinase, methionine adenosyltransferase, and an O-methyltransferase, key components of the methylation pathway. The Δ sahh knockout mutants showed a phenotype of slower growth rate, fewer aerial hyphae, loss of orange pigment, absence of asexual fruiting bodies and conidia, and a significant reduction in virulence. Deletion of sahh significantly reduced the accumulation level of transcripts of the cyp1 that encodes cyclophilin A as well as genes of the heterotrimeric G-protein signaling pathways including cpga1, cpgb1, and cpgc1 and ste12, a target activated by the MAP kinase cascade. Taken together, we demonstrated that SAHH is required for virulence and multiple traits of phenotype in C. parasitica, by regulation of the expression of genes involved in key process of the cell. [ABSTRACT FROM AUTHOR]

Background: Pile fermentation is one of the key steps in developing the Liupao tea (LBT) quality and unique characteristics. The complex biochemical profile of LBT results from microorganisms present during the pile-fermentation process. However, the critical underlying microorganisms and the marker compounds still need to be determined., Results: Staphylococcus, Brevibacterium, Kocuria, Aspergillus, and Blastobotrys were the common dominant microorganisms at the end of the pile fermentation of LBT. Staphylococcus, Aspergillus, Blastobotrys, and nine other genera carried by raw tea are the core microorganisms in the LBT during pile fermentation. A total of 29 critical compounds contributed to the metabolic changes caused by the processing of LBT. Of these, gallic acid, adenine, hypoxanthine, uridine, betaine, 3,4-dihydroxybenzaldehyde, and α-linolenic acid could be characterized as potential marker compounds. Correlation analysis showed that the core microorganisms, including Sphingomonas, Staphylococcus, Kocuria, Aureobasidium, Blastobotrys, Debaryomyce, and Trichomonascus, were closely related to major chemical components and differential compounds. Moreover, the mutually promoting Staphylococcus, Kocuria, Blastobotrys, and Trichomonascus were correlated with the enrichment of marker compounds. Integrated molecular networking and metabolic pathways revealed relevant compounds and enzymes that possibly affect the enrichment of marker compounds., Conclusion: This study analyzed the LBT fermentation samples by omics analysis to reveal the stable microbial community structure, critical microorganisms, and markers compounds affecting the quality of LBT, which contributes to a better understanding of pile fermentation of LBT and the fermentation theory of dark tea. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)