Microscopic green algae are active producers of beneficial compounds, particularly those containing nitrogen. However, the metabolism of nitrogen-containing compounds is diverse and depends on the conditions of the nitrogen source. As a result, the approach to studying the metabolism of nitrogen-containing compounds becomes more complicated. This work demonstrates the metabolic changes in the high-productive green algae Neochlorella semenenkoi IPPAS C-1210 under conditions of nitrogen starvation and subsequent reintake, using high-performance liquid chromatography–mass spectrometry (HPLC–MS) with ¹⁵N isotopic labeling. The presented results include semi-quantitative chromatography–mass spectrometric analysis for 17 amino acids, a metabolomic profile of over 40 isotopically labeled compounds, an assessment of metabolic flux via isotopic incorporation, and an analysis of cellular lipid composition under varying growth conditions. The findings indicate that this strain can utilize ammonium acetate as a nitrogen source, consuming nitrogen in the ammonium form. The degree of isotopic labeling in compounds often diverged significantly from their quantitative changes (concentrations and chromatographic peak areas), suggesting that isotopic analysis may offer advantages over purely quantitative analysis for biological systems. Furthermore, in vivo biological isotopic labeling is shown to assist in identifying compounds absent from standard mass spectrometric databases.

Measurement of the turnover rate of proteins, different metabolites and lipids in living organisms is important for the understanding of biochemical pathways and physiology studies. Such experiments can be performed by administering isotopically labeled substances (food or water) to the organism and measuring the amount of the isotopes in the endogenous compounds. Here, we administered 20% heavy water (D₂O) to a guinea pig for 156 days and regularly measured the deuterium uptake in C-H groups in the different compounds of blood, urine and feces using high-resolution mass spectrometry. We successfully measured the time required for reaching the maximum deuteration level for several classes of compounds: 10 days for blood lipids (PC, PE, TAG); 60 days for sterol derivatives, heme B and hemoglobin; and 70 days for stercobilin. Also, for those compounds, we measured the deuterium elimination time from the organism when deuterium administration was stopped. The turnover of lipids was also studied by administering deuterated oat leaves grown at 10% D₂O to the guinea pig. The analysis of blood revealed that triglycerides demonstrate the inclusion of the deuterium after 5 h. All experiments were performed on a single guinea pig that remained alive and in good health after all experiments. The current research demonstrates the possibility of using long-term D₂O administration for the investigation of metabolism.

The gut microbiota actively metabolizes steroid hormones, but the mechanisms of these transformations—particularly the sequence of enzymatic reactions and the source of hydrogen atoms—remain poorly understood. Conventional analytical approaches are hampered by the complex fecal matrix, isomeric metabolites, and the lack of authentic reference standards. Here we present a strategy based on parallel incubation of steroids with pooled human gut microbiota in H₂O and D₂O, followed by HPLC-HRMS, with the aim of determining the sequence of reductive steps and tracking the incorporation of atoms into steroid metabolites. Using a pooled fecal inoculum from multiple donors (n = 18) and three steroid substrates, we demonstrate that the characteristic mass shift (1.0063 Da per deuterium atom) enables detection of metabolites and distinguishes multi-step enzymatic reactions in the microbial community without recombinant enzymes and authentic standards. Progesterone and 19-hydroxy-4-androstene-3,17-dione underwent sequential two-step reduction incorporating up to three deuterium atoms, while 17α-hydroxypregnenolone followed a three-step pathway incorporating up to four deuterium atoms, consistent with localization to the A-ring. This workflow provides a practical tool for investigating gut microbial steroid metabolism in the context of human physiology and disease, with potential applications in monitoring microbial activity in endocrine disorders, inflammatory bowel disease, and neuropsychiatric conditions.

Microalgae of the Chlorellaceae family are promising producers of valuable metabolites, yet the choice of nitrogen source significantly modulates their physiology and biochemistry. In this study, we investigated the effect of two inorganic nitrogen sources—ammonium acetate (AA) and sodium nitrate—on the metabolic recovery of Neochlorella semenenkoi IPPAS C-1210 after nitrogen starvation. Using a combination of targeted amino acid quantification (HPLC-MS/MS) and untargeted high-resolution metabolomics, we tracked both concentration changes and de novo biosynthesis of nitrogen-containing metabolites via ¹⁵N isotopic labeling. Our results show that ammonium acetate and sodium nitrate enable equally rapid growth resumption and comparable high final biomass yields. Targeted analysis revealed that ammonium-fed cells restored their amino acid pools substantially within 1 day, with a pronounced accumulation of glutamate and glutamine, whereas nitrate-fed cells exhibited a slower and only partial recovery of their free amino acid pool, with several amino acids remaining undetectable even after 3 days. Untargeted metabolomics identified 308 unique metabolites, and the heatmaps of the most variable compounds highlighted a marked early upregulation of TCA cycle intermediates, stress-signaling molecules, and nucleotide precursors in AA cultures, in contrast to a delayed, lipid-centric response in nitrate cultures. The ¹⁵N tracing confirmed near-complete (>96%) enrichment of amino acids in both sources, demonstrating that recovery is driven by de novo synthesis rather than recycling, and that isotopic enrichment extends to nucleotides and the NAD⁺ precursor nicotinamide. These findings reveal the metabolic advantages of ammonium over nitrate for rapid metabolic recovery and restoration of nitrogen-rich compound pools, and provide a foundation for optimizing cultivation strategies for this biotechnologically relevant alga.