Summary of Study ST004088
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002567. The data can be accessed directly via it's Project DOI: 10.21228/M8TV7M This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004088 |
| Study Title | Maternal Vitamin D Status Influences Neuroactive Metabolites in Breast Milk and Shapes the Infant Gut Metabolome |
| Study Type | Nutritional biochemistry-based clinical trial |
| Study Summary | This study is the first to demonstrate that maternal vitamin D status influences the levels of neurotransmitter precursors, such as tryptophan, phenylalanine, and tyrosine, in human breast milk and stool metabolic profiles in infants. These findings highlight the potential impact of maternal nutrition on early-life gut metabolome development, emphasizing the broader implications of maternal vitamin D status for infant health and neurodevelopment. |
| Institute | Medical University of South Carolina |
| Department | Regenerative & Cellular Medicine |
| Laboratory | Wagner Laboratory |
| Last Name | Carol |
| First Name | Wagner |
| Address | 96 Jonathan Lucas St. Ste. 601, MSC 617, Charleston, SC 29425 |
| wagnercl@musc.edu | |
| Phone | 843-792-8892 |
| Submit Date | 2025-07-28 |
| Num Groups | 2 cohorts |
| Total Subjects | 6 subjects |
| Num Males | for the infant stool portion of the study, 3 male subjects |
| Num Females | for the breast milk portion of the study, 6 female subjects; for the infant stool portion of the study, 3 female subjects |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2026-01-02 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002567 |
| Project DOI: | doi: 10.21228/M8TV7M |
| Project Title: | Maternal Vitamin D Status Influences Neuroactive Metabolites in Breast Milk and Shapes the Infant Gut Metabolome |
| Project Type: | Basic Research - Nutrition in Pregnancy Study |
| Project Summary: | Human milk contains multiple bioactive components, many of which are influenced by the mother's nutritional status. Previous research has demonstrated that adequate maternal vitamin D levels can influence the protein, lipid, microbial, and immunological profiles of breast milk. However, the impact of maternal vitamin D status on neurotransmitters in breast milk and the infant gut remains largely unexplored. We conducted a post-hoc analysis using breast milk and matched infant stool samples from a 3-month randomized controlled trial where exclusively breastfeeding mothers were examined for vitamin D levels and categorized as sufficient or deficient for vitamin D. Neuroactive metabolites and short-chain fatty acids (SCFAs) were quantified using both targeted and non-targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS). Our findings revealed that breast milk from mothers with sufficient vitamin D levels contained significantly higher concentrations of tryptophan, phenylalanine, and tyrosine compared to milk from mothers with lower vitamin D levels. No significant differences were observed in tryptamine, kynurenine, kynurenic acid, anthranilic acid, quinolinic acid, tyramine, dopamine, epinephrine, or norepinephrine between the two groups. Among SCFAs, only hexanoic acid was significantly elevated in the breast milk of mothers with sufficient vitamin D. A non-targeted metabolomics analysis of infant stool identified distinct metabolite profiles, where oleamide, vaccenic acid, lacto-N-triaose, and N-acetyl-D-glucosamine varied according to maternal vitamin D levels, indicating that maternal nutrient status may influence the infant gut metabolome. These findings suggest that maternal vitamin D status can influence neurotransmitter precursor levels in breast milk and alter the metabolomic profile of infant stool. |
| Institute: | Baylor College of Medicine |
| Department: | Pathology & Immunology |
| Laboratory: | Horvath Lab |
| Last Name: | Horvath |
| First Name: | Thomas |
| Address: | 1 Baylor Plaza, Houston, TX, 77030, USA |
| Email: | thomas.horvath2@bcm.edu |
| Phone: | 832-824-0904 |
| Funding Source: | T32DK124191-01A1, K01DK123195, P30 DK123704, P20 GM120457, NATS NIH KL2TR001452, UL1TR001450, S10OD036416, P30 DK056338 |
| Publications: | American Journal of Physiology - Gastrointestinal & Liver submission is currently under review |
| Contributors: | Alyssa S. Gutierrez1, Katherine E. Chetta2,3, Santosh Thapa4, Sigmund J. Haidacher5,6, Kathleen M. Hoch5,6, John E. Baatz2,3, Anthony M. Haag5,6, Numan Oezguen5,6, Thomas D. Horvath5,6,7, Carol L. Wagner¥2,3, Melinda A. Engevik¥5,8 |
Subject:
| Subject ID: | SU004234 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | N/A |
| Age Or Age Range: | We are blinded to this information |
| Weight Or Weight Range: | We are blinded to this information |
| Height Or Height Range: | We are blinded to this information |
| Gender: | Male and female |
| Human Race: | We are blinded to this information |
| Human Ethnicity: | We are blinded to this information |
| Human Trial Type: | Basic Research - Nutrition in Pregnancy |
| Human Lifestyle Factors: | We are blinded to this information |
| Human Medications: | We are blinded to this information |
| Human Prescription Otc: | We are blinded to this information |
| Human Smoking Status: | We are blinded to this information |
| Human Alcohol Drug Use: | We are blinded to this information |
| Human Nutrition: | New mother were randomized to either a placebo group, or a vitamin-D3 supplemented group. |
| Human Inclusion Criteria: | Mother's need to have infants born > 35 weeks gestation, be in good health, and be exclusively breastfeeding |
| Human Exclusion Criteria: | non-breastfeeding; we are blinded to other potential exclusion criteria |
| Species Group: | Homo sapiens |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Vitamin D Status | Sample source |
|---|---|---|---|
| SA473901 | Pos_S5 | High | Infant Stool |
| SA473902 | Neg_S6 | High | Infant Stool |
| SA473903 | Neg_S4 | High | Infant Stool |
| SA473904 | Neg_S5 | High | Infant Stool |
| SA473905 | Pos_S6 | High | Infant Stool |
| SA473906 | Pos_S4 | High | Infant Stool |
| SA473907 | M6 | High | Maternal Breast Milk |
| SA473908 | M4 | High | Maternal Breast Milk |
| SA473909 | M5 | High | Maternal Breast Milk |
| SA473910 | Pos_S1 | Low | Infant Stool |
| SA473911 | Pos_S3 | Low | Infant Stool |
| SA473912 | Pos_S2 | Low | Infant Stool |
| SA473913 | Neg_S3 | Low | Infant Stool |
| SA473914 | Neg_S1 | Low | Infant Stool |
| SA473915 | Neg_S2 | Low | Infant Stool |
| SA473916 | M1 | Low | Maternal Breast Milk |
| SA473917 | M3 | Low | Maternal Breast Milk |
| SA473918 | M2 | Low | Maternal Breast Milk |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO004227 |
| Collection Summary: | Given that the authors still are blinded to assigned study groups from the larger clinical trial, the samples were selected based on 3-month circulating maternal vitamin D levels and were not directly related to the supplementation in the larger clinical trial. A total of six mother-child dyads were selected based on serum vitamin D levels. According to the NIH, vitamin D sufficiency can be confirmed by serum 25-D concentrations of ≥20 ng/mL (50 nmol/L) (35). Three dyads were selected by sufficient maternal serum vitamin D (25-D >45 ng/mL), while the other three dyads were chosen by deficient maternal serum vitamin D (25-D <20 ng/mL). The mothers were all in good general health and the infants were all between the ages of 1-3 months. At the three-month follow up visit, the mother expressed breast milk in a hygienic fashion into a container as per study protocol, and an infant stool was collected from a diaper. Samples were freshly collected in the early morning and stored during the AM research clinic visit. Breast milk (n=6) and stool samples (n=6) were collected from mother-child dyads. We analyzed feces from 2 female and 1 male infant from mothers with sufficient vitamin D and 2 male and 1 female infants from mothers with insufficient vitamin D status. |
| Collection Protocol ID: | This study was approved by the Medical University of South Carolina (PRO #0050609), and the methods have been previously reported |
| Sample Type: | Milk |
Treatment:
| Treatment ID: | TR004243 |
| Treatment Summary: | Exclusively breastfeeding mothers in good general health and their infants (≥ 35 weeks’ gestation) were enrolled in the parent trial (6), and written consent was obtained for participation. In this study, mothers were randomized to either placebo or 400, 2400, or 6400 IU vitamin D3/day for 6 months. No other vitamins were supplemented. |
Sample Preparation:
| Sampleprep ID: | SP004240 |
| Sampleprep Summary: | All samples were processed using ice cold methanol. Breast milk samples were treated with an equal volume of ice-cold methanol (1:1 v/v methanol). Tubes were centrifuged at 12,000 x g for 5 min to pellet any solids and the cell-free supernatant was used for downstream analysis. Infant stool samples were weighed and 100 mg of feces was added into 2 mL fast prep-tubes (MP Biomedical #MP115076200) harboring 0.1 g of 1.4 mm ceramic beads (MP Biomedical Lysing Matrix D #116540434); similar to previously published work (37). To each tube, a 1 mL volume of ice-cold methanol and water (1:1, v/v) was added and the tubes were homogenized on a Benchmark “Beadbug” 6 Microtube Homogenizer (Stellar Scientific, #BS-BEBU-6) at 4.0 m/s for 20 seconds for two cycles. Tubes were centrifuged at 12,000 x g for 5 min to pellet any solids and cell-free supernatant was used for downstream analysis. |
| Processing Storage Conditions: | On ice |
Combined analysis:
| Analysis ID | AN006776 | AN006777 | AN006778 |
|---|---|---|---|
| Chromatography ID | CH005146 | CH005146 | CH005146 |
| MS ID | MS006475 | MS006476 | MS006477 |
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Shimadzu 20AD | Shimadzu 20AD | Shimadzu 20AD |
| Column | Phenomenex Luna C18 (100 mm x 1 mm, 3 µm, 100 angstrom Pore) | Phenomenex Luna C18 (100 mm x 1 mm, 3 µm, 100 angstrom Pore) | Phenomenex Luna C18 (100 mm x 1 mm, 3 µm, 100 angstrom Pore) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Fisher Scientific Orbitrap Fusion | Thermo Fisher Scientific Orbitrap Fusion | Thermo Fisher Scientific Orbitrap Fusion |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE |
| Units | Peak Area (Ion Counts) | Peak Area (Ion Counts) | Peak Area (Ion Counts) |
Chromatography:
| Chromatography ID: | CH005146 |
| Chromatography Summary: | The reverse-phase chromatographic separation for the non-targeted metabolomics measurement was performed using a Luna C18(2) and Security Guard C18 analytical and guard column combination described above, and using a MPA solution consisting of water that contained 0.1% formic acid, a MPB solution consisting of acetonitrile that contained 0.1% formic acid, and a NW solution composed of a mixture of acetonitrile:Water (1:1, v:v). The mobile phase flowrate was 0.080 mL/min, the autosampler tray was chilled to 15°C, and the column oven was heated to 40°C. The gradient elution program was specified as follows: 0-2 min, 5% MPB; 2-25 min, 5-90% MPB; 25-30 min, 90% MPB; 30-31 min, 90-5% MPB; and, 31-40 min, 5% MPB; and at 40.01 min a Stop command is specified, and there is a 40.4 min duty cycle for each injection. |
| Instrument Name: | Shimadzu 20AD |
| Column Name: | Phenomenex Luna C18 (100 mm x 1 mm, 3 µm, 100 angstrom Pore) |
| Column Temperature: | 40°C |
| Flow Gradient: | 0-2 min, 5% B; 2-25 min, 5-90% B; 25-30 min, 90% B; 30-31 min, 90-5% B; and, 31-40 min, 5% B |
| Flow Rate: | 0.080 mL/min |
| Solvent A: | 100% Water; 0.1% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1% Formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS006475 |
| Analysis ID: | AN006776 |
| Instrument Name: | Thermo Fisher Scientific Orbitrap Fusion |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Non-targeted metabolomics experiments were performed using positive mode acquisitions for maternal breast milk sample extracts. The heated electrospray (H-ESI) ionization source parameters for the Orbitrap Fusion MS were specified in the Xcalibur-based acquisition method as follows: application mode: small molecule; method duration: 39.5 minutes; ionization mode polarity, positive mode and negative mode; probe spray voltage, static at +3,500 V (positive mode); gas mode, static; sheath gas (arbitrary units), 25; auxiliary gas (arbitrary units), 5; sweep gas (arbitrary units), 0; ion transfer tube temperature, 275°C; and, vaporizer temperature, 75°C. Global MS acquisition parameters were specified as follows: infusion mode, liquid chromatography; expected LC peak width (s), 30; advanced peak determination, false; mild trapping, false; default charge state, 1; enable Xcalibur AcquireX method modification, false; and, internal mass calibration, Easy-IC™. The acquisition parameters for the HRMS/HRMS acquisition method used for the reverse-phase chromatographic method, for both positive and negative mode ionization methods, was specified as follows: detector type, Orbitrap; Orbitrap resolution, 120,000 (at m/z 200); mass range, normal; use quadrupole isolation, true; scan range (m/z), 100-650; RF lens (%), 60; AGC target, standard; maximum injection time mode, custom; maximum injection time (ms), 50; Microscans, 1; data type, profile; polarity, positive and negative for each method; source fragmentation, disabled; and, use Easy-IC™, true. Dynamic exclusion filtering was enabled with the following parameters specified: exclude after n times, 1; exclusion duration (s), 6; mass tolerance, ppm; low, 10; high, 10; exclude isotopes, true; perform dependent scan on single charge state per precursor only, false; and, exclude within cycle, true. Apex detection filtering was enabled with the following parameters specified: expected peak width (FWHM, s), 10; desired apex window (%), 30. Intensity threshold filtering was enabled with the following parameters specified: filter type, intensity threshold; intensity threshold, 5.0e4. The ddMS2 OT HCD based MS2 scan function was enabled with the following parameters specified: isolation mode, quadrupole; isolation window (m/z), 2; isolation offset, off; activation type, HCD; collision energy mode, stepped; HCD collision energies (%), 15, 25, and 35; detector type, Orbitrap; Orbitrap resolution, 30,000 (at m/z 200); mass range, normal; scan range mode, auto; AGC target, standard; maximum injection time mode, custom; maximum injection time, 54 ms; Microscans, 1; data type, profile; and, Use Easy-IC™, true. |
| Ion Mode: | POSITIVE |
| MS ID: | MS006476 |
| Analysis ID: | AN006777 |
| Instrument Name: | Thermo Fisher Scientific Orbitrap Fusion |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Non-targeted metabolomics experiments were performed using negative mode acquisitions for infant fecal sample extracts. The heated electrospray (H-ESI) ionization source parameters for the Orbitrap Fusion MS were specified in the Xcalibur-based acquisition method as follows: application mode: small molecule; method duration: 39.5 minutes; ionization mode polarity, positive mode and negative mode; probe spray voltage, static at -2,500 V (negative mode); gas mode, static; sheath gas (arbitrary units), 25; auxiliary gas (arbitrary units), 5; sweep gas (arbitrary units), 0; ion transfer tube temperature, 275°C; and, vaporizer temperature, 75°C. Global MS acquisition parameters were specified as follows: infusion mode, liquid chromatography; expected LC peak width (s), 30; advanced peak determination, false; mild trapping, false; default charge state, 1; enable Xcalibur AcquireX method modification, false; and, internal mass calibration, Easy-IC™. The acquisition parameters for the HRMS/HRMS acquisition method used for the reverse-phase chromatographic method, for both positive and negative mode ionization methods, was specified as follows: detector type, Orbitrap; Orbitrap resolution, 120,000 (at m/z 200); mass range, normal; use quadrupole isolation, true; scan range (m/z), 100-650; RF lens (%), 60; AGC target, standard; maximum injection time mode, custom; maximum injection time (ms), 50; Microscans, 1; data type, profile; polarity, positive and negative for each method; source fragmentation, disabled; and, use Easy-IC™, true. Dynamic exclusion filtering was enabled with the following parameters specified: exclude after n times, 1; exclusion duration (s), 6; mass tolerance, ppm; low, 10; high, 10; exclude isotopes, true; perform dependent scan on single charge state per precursor only, false; and, exclude within cycle, true. Apex detection filtering was enabled with the following parameters specified: expected peak width (FWHM, s), 10; desired apex window (%), 30. Intensity threshold filtering was enabled with the following parameters specified: filter type, intensity threshold; intensity threshold, 5.0e4. The ddMS2 OT HCD based MS2 scan function was enabled with the following parameters specified: isolation mode, quadrupole; isolation window (m/z), 2; isolation offset, off; activation type, HCD; collision energy mode, stepped; HCD collision energies (%), 15, 25, and 35; detector type, Orbitrap; Orbitrap resolution, 30,000 (at m/z 200); mass range, normal; scan range mode, auto; AGC target, standard; maximum injection time mode, custom; maximum injection time, 54 ms; Microscans, 1; data type, profile; and, Use Easy-IC™, true. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS006477 |
| Analysis ID: | AN006778 |
| Instrument Name: | Thermo Fisher Scientific Orbitrap Fusion |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Non-targeted metabolomics experiments were performed using positive mode acquisitions for infant fecal sample extracts. The heated electrospray (H-ESI) ionization source parameters for the Orbitrap Fusion MS were specified in the Xcalibur-based acquisition method as follows: application mode: small molecule; method duration: 39.5 minutes; ionization mode polarity, positive mode and negative mode; probe spray voltage, static at +3,500 V (positive mode); gas mode, static; sheath gas (arbitrary units), 25; auxiliary gas (arbitrary units), 5; sweep gas (arbitrary units), 0; ion transfer tube temperature, 275°C; and, vaporizer temperature, 75°C. Global MS acquisition parameters were specified as follows: infusion mode, liquid chromatography; expected LC peak width (s), 30; advanced peak determination, false; mild trapping, false; default charge state, 1; enable Xcalibur AcquireX method modification, false; and, internal mass calibration, Easy-IC™. The acquisition parameters for the HRMS/HRMS acquisition method used for the reverse-phase chromatographic method, for both positive and negative mode ionization methods, was specified as follows: detector type, Orbitrap; Orbitrap resolution, 120,000 (at m/z 200); mass range, normal; use quadrupole isolation, true; scan range (m/z), 100-650; RF lens (%), 60; AGC target, standard; maximum injection time mode, custom; maximum injection time (ms), 50; Microscans, 1; data type, profile; polarity, positive and negative for each method; source fragmentation, disabled; and, use Easy-IC™, true. Dynamic exclusion filtering was enabled with the following parameters specified: exclude after n times, 1; exclusion duration (s), 6; mass tolerance, ppm; low, 10; high, 10; exclude isotopes, true; perform dependent scan on single charge state per precursor only, false; and, exclude within cycle, true. Apex detection filtering was enabled with the following parameters specified: expected peak width (FWHM, s), 10; desired apex window (%), 30. Intensity threshold filtering was enabled with the following parameters specified: filter type, intensity threshold; intensity threshold, 5.0e4. The ddMS2 OT HCD based MS2 scan function was enabled with the following parameters specified: isolation mode, quadrupole; isolation window (m/z), 2; isolation offset, off; activation type, HCD; collision energy mode, stepped; HCD collision energies (%), 15, 25, and 35; detector type, Orbitrap; Orbitrap resolution, 30,000 (at m/z 200); mass range, normal; scan range mode, auto; AGC target, standard; maximum injection time mode, custom; maximum injection time, 54 ms; Microscans, 1; data type, profile; and, Use Easy-IC™, true. |
| Ion Mode: | POSITIVE |
