Data from: A simple, anesthesia-free infusion technique for in vivo metabolic tracing
Data files
Jul 31, 2026 version files 643.30 KB
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README.md
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Source_data_Figure_1.xlsx
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Source_data_Figure_2.xlsx
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Source_data_Figure_3.xlsx
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Source_data_Figure_4.xlsx
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Source_data_Supplementary_Figure_1.xlsx
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Source_data_Supplementary_Figure_2.xlsx
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Source_data_Supplementary_Figure_3.xlsx
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Source_data_Supplementary_Figure_4.xlsx
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Abstract
Accurate metabolic flux analysis requires tracer delivery that preserves physiological metabolism. Current methods may distort metabolism through isoflurane anesthesia, surgical stress, or complex procedures. We demonstrate that isoflurane anesthesia profoundly alters serum and tissue metabolism across multiple pathways. In serum, acylcarnitines and fatty acids were broadly decreased, while amino acid metabolites and select nucleotide species were increased. Across multiple organs, isoflurane induced coordinated metabolic remodeling and distinct tissue-specific responses, including glycolytic remodeling in the brain and amino acid accumulation in the pancreas. To address these metabolic disturbances, we established a non-surgical tail vein catheterization method completed in minutes under brief isoflurane anesthesia that enables multi-hour tracer infusion in awake, freely moving mice. Using U-13C6-cystine infusion, this method achieved robust cystine labeling and downstream labeling comparable to jugular infusion while maintaining circulating cystine pools closer to physiological levels. This platform provides a practical approach for in vivo stable isotope tracing under more physiological conditions.
Dataset DOI: 10.5061/dryad.zw3r228nd
Description of the data and file structure
This dataset contains source data supporting the figures in the manuscript “A simple, anesthesia-free infusion technique for in vivo metabolic tracing.”
Files are organized by figure number (Source_data_Figure_1-4.xlsx, Source_data_Supplementary_Figure_1-4.xlsx) and contain the quantitative values used to generate the corresponding plots.
Note on missing values: Cells marked "n/a" indicate no data for that replicate position, either because experimental groups had unequal sample sizes or because a given isotopologue was not detected/applicable for that metabolite. A value of 0 reflects an actual measurement at or below the detection limit and is distinct from "n/a."
Files and variables
File: Source_data_Figure_1.xlsx
Description: This Excel file contains the source data used to generate Figure 1, which characterizes global serum metabolomic changes following 2 h isoflurane anesthesia compared with anesthesia-free controls. The workbook contains data for Figure 1B (summary of significantly altered metabolites by biochemical category) and Figure 1C–F (dot-heatmap of metabolite changes across biochemical categories).
Variables
Figure 1B: A total of 265 metabolites were detected in serum; of these, 23 were significantly increased and 15 were significantly decreased in the anesthesia group (fold change ≥ 2, P < 0.05).
- Classification: Biochemical metabolite category (Central Energy Metabolism, Amino Acid Metabolism, Lipid Metabolism, Others).
- Increased metabolites: Number of metabolites significantly increased in the anesthesia group.
- Increased %: Percentage of the 265 detected metabolites that were significantly increased within each category.
- Decreased metabolites: Number of metabolites significantly decreased in the anesthesia group.
- Decreased %: Percentage of the 265 detected metabolites that were significantly decreased within each category.
Figure 1C-F
- Compound: Metabolite name.
- Awake_[Sex][Number]: Raw LC-MS abundance values for individual anesthesia-free control male (M) and female (F) mice (n = 5 per sex).
- Anesthesia_[Sex][Number]: Raw LC-MS abundance values for individual isoflurane-anesthetized male (M) and female (F) mice (n = 5 per sex).
- P-value: Statistical significance for the comparison between groups (unpaired Student's t-test).
- Log(FC): Log2 fold change of anesthesia versus anesthesia-free abundance.
- Main Class: Primary biochemical pathway annotation.
- Sub Class: Secondary biochemical pathway annotation.
File: Source_data_Figure_2.xlsx
Description: This Excel file contains the source data used to generate Figure 2, which characterizes tissue-level metabolomic changes in brain, lung, liver, and pancreas following 2 h isoflurane anesthesia. The workbook contains data for Figure 2A–D (volcano plots of global metabolomics per tissue) and Figure 2E–F (dot-heatmaps of metabolic changes grouped by biochemical pathway across tissues).
Variables
Figure 2A (Brain), 2B (Lung), 2C (Liver), 2D (Pancreas)
- compound in [Tissue]: Metabolite name.
- Awake_[Sex][Number]: Raw LC-MS abundance values for individual anesthesia-free male (M) and female (F) mice. Brain (2A) has n = 4; Liver, Lung, and Pancreas (2B–2D) have n = 5.
- Anesthesia_[Sex][Number]: Raw LC-MS abundance values for individual isoflurane-anesthetized male (M) and female (F) mice (n = 5 per sex).
- Log(p-value): Log10-transformed P-value for the comparison between groups.
- Log(FC): Log2 fold change of anesthesia versus anesthesia-free abundance.
Figure 2E-F
This sheet has a two-row header (tissue name, then statistic) reflecting comparisons made separately for each of the four tissues.
- Metabolites: Metabolite name.
- Brain / Liver / Lung / Pancreas – p-value: Statistical significance for the comparison between groups in that tissue.
- Brain / Liver / Lung / Pancreas – Log(FC): Log2 fold change in that tissue.
- Classification: Primary biochemical pathway annotation.
- Sub_Classification: Secondary biochemical pathway annotation.
File: Source_data_Figure_3.xlsx
Description: This Excel file contains the source data used to generate Figure 3, which compares jugular and tail vein catheterization for U-¹³C₆-cystine infusion. The workbook contains data for Figure 3E (serum corticosterone levels across infusion groups) and Figure 3F (total serum cystine pool sizes showing unlabeled and ¹³C-labeled fractions).
Variables
Figure 3E:
- Tail / Jugular / No Infusion: Serum corticosterone abundance (LC-MS peak area) for individual mice in each infusion group (Tail n = 13, Jugular n = 12, No Infusion n = 8).
Figure 3F
- Group: Infusion group assignment (Tail, Jugular, No Infusion).
- 13C labeling: Isotopologue label state — "Unlabeled" (M+0) or "13C-labeled" (M+3 and M+6 combined).
- Mouse1–Mouse13: Cystine abundance (LC-MS peak area) for each individual mouse (Tail n = 13, Jugular n = 12, No Infusion n = 8).
File: Source_data_Figure_4.xlsx
Description: This Excel file contains the source data used to generate Figure 4, which presents isotopologue tracing of U-¹³C₆-cystine incorporation into downstream sulfur metabolites. The workbook contains data for Figure 4B (serum cystine isotopologue distribution), Figure 4C (isotopologue fractions of cystine-derived metabolites in pancreas), and Figure 4D (isotopologue fractions in kidney).
Variables
Figure 4B (Serum)
- Metabolite: Cystine (CYS2).
- Group: Infusion route (Tail, Jugular).
- Isotopologue: M+0, M+3, or M+6.
- Mouse1–Mouse12: Isotopologue fraction for each individual mouse (Tail n = 9, Jugular n = 12).
Figure 4C (Pancreas) and Figure 4D (Kidney)
- Tissue: Pancreas or Kidney.
- Metabolite: CYS2 (cystine), CYS (cysteine), GLU-CYS (γ-glutamylcysteine), GSH (glutathione), HTAU (hypotaurine), or TAU (taurine).
- Group: Infusion route (Tail, Jugular).
- Isotopologue: M+0, M+3, or M+6. Not every metabolite shows detectable labeling at every isotopologue.
- Mouse1–Mouse12: Isotopologue fraction for each individual mouse (Tail n = 9, Jugular n = 12).
File: Source_data_Supplementary_Figure_1.xlsx
Description: This Excel file contains the source data used to generate Supplementary Figure 1, which reports serum corticosterone levels following 2 h isoflurane anesthesia. The workbook contains data for Supplementary Figure 1A (total serum corticosterone in anesthesia-free control versus isoflurane anesthesia groups) and Supplementary Figure 1B (sex-stratified analysis).
Variables
Supplementary Figure 1A
- Control: Serum corticosterone abundance for anesthesia-free control mice.
- Anesthesia: Serum corticosterone abundance for isoflurane-anesthetized mice.
Supplementary Figure 1B:
Rows give the experimental group (Control, Anesthesia).
- male1–male5: Serum corticosterone abundance for individual male mice in that group.
- Female1–Female5: Serum corticosterone abundance for individual female mice in that group.
File: Source_data_Supplementary_Figure_2.xlsx
Description: This Excel file contains the source data used to generate Supplementary Figure 2, which demonstrates that brief isoflurane exposure and tail-vein catheter implantation do not induce persistent metabolomic alterations. The workbook contains data for Supplementary Figure 2A–B (PCA and hierarchical clustering heatmap of serum metabolomic profiles across four experimental groups) and Supplementary Figure 2C (total serum corticosterone across groups).
Variables
Supplementary Figure 2A–B
- Metabolite: Metabolite name.
- Brief Isoflurane1–8: Raw LC-MS abundance for individual mice in the brief isoflurane group (n = 8).
- Catheter implantation1–9: Raw LC-MS abundance for individual mice in the catheter implantation group (n = 9).
- Control1–10: Raw LC-MS abundance for individual mice in the control group (n = 10).
- Isoflurane(2h)1–10: Raw LC-MS abundance for individual mice in the 2 h isoflurane group (n = 10).
- FDR: False discovery rate from one-way ANOVA across the four groups; metabolites shown in the heatmap were selected at FDR < 0.01.
Supplementary Figure 2C
- Control / Brief Isoflurane / Catheter implantation / Isoflurane(2h): Serum corticosterone abundance for individual mice in each group.
File: Source_data_Supplementary_Figure_3.xlsx
Description: This Excel file contains the source data used to generate Supplementary Figure 3, which presents normalized isotopologue enrichment and total metabolite pool sizes for cystine-derived metabolites in pancreas and kidney following jugular or tail-vein U-¹³C₆-cystine infusion. The workbook contains data for Supplementary Figure 3A (isotopologue fractions normalized to tissue ¹³C-cystine enrichment) and Supplementary Figure 3B (absolute pool sizes of unlabeled and labeled isotopologues).
Variables
Supplementary Figure 3A
- Tissue: Pancreas or Kidney.
- Metabolite: CYS2 (cystine), CYS (cysteine), GLU-CYS (γ-glutamylcysteine), GSH (glutathione), HTAU (hypotaurine), or TAU (taurine).
- Group: Infusion route (Tail n = 9, Jugular n = 12).
- Isotopologue: M+3 or M+6.
- Mouse1–Mouse12: Isotopologue fraction for each individual mouse.
Supplementary Figure 3B
- Tissue: Pancreas or Kidney.
- Metabolite: CYS2 (cystine), CYS (cysteine), GLU-CYS (γ-glutamylcysteine), GSH (glutathione), HTAU (hypotaurine), or TAU (taurine).
- Group: Infusion route (Tail n = 9, Jugular n = 12).
- Isotopologue: M+0, M+3, or M+6.
- Mouse1–Mouse12: Absolute LC-MS peak area for each individual mouse.
File: Source_data_Supplementary_Figure_4.xlsx
Description: This Excel file contains the source data used to generate Supplementary Figure 4, which compares awake and isoflurane-anesthetized conditions during tail-vein U-¹³C₆-cystine infusion. The workbook contains data for Supplementary Figure 4A (serum cystine isotopologue distributions), Figure 4B (isotopologue distributions of cystine-derived metabolites in pancreas and kidney), Supplementary Figure 4C (total serum cystine pool sizes), and Supplementary Figure 4D (total metabolite pool sizes in pancreas and kidney).
Variables
Supplementary Figure 4A and 4C (Serum)
- Metabolite: Cystine (CYS2).
- Group: Infusion condition (Awake n = 7, Asleep n = 9).
- Isotopologue: M+0, M+3, or M+6.
- Mouse1–Mouse9: Isotopologue fraction (4A) or absolute peak area (4C) for each individual mouse.
Supplementary Figure 4B and 4D (Pancreas and Kidney)
- Tissue: Pancreas or Kidney.
- Metabolite: CYS2 (cystine), CYS (cysteine), GLU-CYS (γ-glutamylcysteine), GSH (glutathione), HTAU (hypotaurine), or TAU (taurine).
- Group: Infusion condition (Awake n = 7, Asleep n = 9).
- Isotopologue: M+0, M+3, or M+6.
- Mouse1–Mouse9: Isotopologue fraction (4B) or absolute peak area (4D) for each individual mouse.
Code/software
All source data are provided as Microsoft Excel (.xlsx) files and can be viewed using standard spreadsheet software (e.g., Microsoft Excel).
Access information
Other publicly accessible locations of the data:
- An earlier version of the Figure 1 and 2 source data was shared as supplementary tables in the associated bioRxiv preprint. In those preprint experiments, mouse body temperature was not actively controlled during isoflurane anesthesia. The Figure 1 and 2 data deposited here were generated from newly performed experiments, conducted after the preprint was posted, in which mouse body temperature was maintained using a heating pad throughout anesthesia. The experimental concept and figure structure are unchanged, but the two datasets come from distinct experimental cohorts under different temperature conditions and are not directly comparable. No other data associated with this study are publicly available elsewhere.
Data was derived from the following sources:
- All data were generated for this study. The datasets provided here via Dryad represent the final versions of all source data and have not been previously deposited in any public repository. The Figure 1 and 2 source data in the bioRxiv preprint were obtained without active temperature control during anesthesia; the corresponding data deposited here were newly generated with body temperature maintained via heating pad, and therefore reflect a separate experimental cohort rather than a reanalysis of the preprint data.