Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps—from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

Streptomyces venezuelae ATCC 10712 produces antibiotics chloramphenicol (Cml) and jadomycin (Jad) in response to nutrient limitation and ethanol shock (ES), respectively. Biosynthesis of Cml and Jad was shown to be reciprocally regulated via the action of regulatory proteins JadR1 and JadR2 encoded by the jad cluster, and mechanism of such regulation has been characterized. However, detailed analysis of the regulatory mechanism controlling Cml biosynthesis is still lacking. In the present study, several promoters from the cml cluster were fused to the reporter gene gusA. Reporter protein activity and Cml production were assayed in the wild-type strain with and without ES, followed by similar experiments with the jadR1 deletion mutant. The latter gene was earlier reported to negatively control Cml biosynthesis, while serving as a positive regulator for the jad cluster. A double deletion mutant deficient in both jadR1 and the cml cluster was also constructed and used in promoter fusion studies. Analyses of the results revealed that ES activates Cml biosynthesis in both wild-type and jadR1 deletion mutant, while Cml production by the latter was ca 80 % lower. These results contradict earlier reports regarding the function of JadR1, but correlate well with the reporter activity data for some promoters, while reaction of others to the ES is genotype-dependent. Remarkably, the absence of Cml production in the double mutant has a profound effect on the way certain cml promoters react to ES. The latter suggests direct involvement of Cml in this complex regulatory mechanism.

A challenge in formulating water-in-oil-in-water (W/O/W) emulsions is the uncontrolled release of the encapsulated compound prior to application. Pharmaceuticals and nutraceuticals usually have amphipathic nature, which may contribute to leakage of the active ingredient. In the present study, cyclodextrins (CyDs) were used to impart a change in the relative polarity and size of a model compound (ibuprofen) by the formation of inclusion complexes. Various inclusion complexes (2-hydroxypropyl (HP)-β-CyD-, α-CyD- and γ-CyD-ibuprofen) were prepared and presented within W/O/W emulsions, and the initial and long-term encapsulation efficiency was investigated. HP-β-CyD-ibuprofen provided the highest encapsulation of ibuprofen in comparison to a W/O/W emulsion with unassociated ibuprofen confined within the inner water phase, with a four-fold increase in the encapsulation efficiency. An improved, although lower, encapsulation efficiency was obtained for the inclusion complex γ-CyD-ibuprofen in comparison to HP-β-CyD-ibuprofen, whereas α-CyD-ibuprofen had a similar encapsulation efficiency to that of unassociated ibuprofen. The lower encapsulation efficiency of ibuprofen in combination with α-CyD and γ-CyD was attributed to a lower association constant for the γ-CyD-ibuprofen inclusion complex and the ability of α-CyD to form inclusion complexes with fatty acids. For the W/O/W emulsion prepared with HP-β-CyD-ibuprofen, the highest encapsulation of ibuprofen was obtained at hyper- and iso-osmotic conditions and by using an excess molar ratio of CyD to ibuprofen. In the last part of the study, it was suggested that the chemical modification of the HP-β-CyD molecule did not influence the encapsulation of ibuprofen, as a similar encapsulation efficiency was obtained for an inclusion complex prepared with mono-1-glucose-β-CyD.

Background: Nicotinamide adenine dinucleotide (NAD⁺), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD⁺ derivatives and it’s reduced forms, with ¹³C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD⁺ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD⁺ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD⁺ metabolism and the potential significance of these metabolites in various biological contexts.