#METABOLOMICS WORKBENCH dwb7_20230831_114518 DATATRACK_ID:4277 STUDY_ID:ST002937 ANALYSIS_ID:AN004820 PROJECT_ID:PR001827 VERSION 1 CREATED_ON October 12, 2023, 4:34 pm #PROJECT PR:PROJECT_TITLE Deep Metabolic Phenotyping of Newborn Cord Blood Reveals Maternal-Fetal PR:PROJECT_TITLE Interactions and Disease Risk PR:PROJECT_SUMMARY Metabolites are small molecules circulating in the mother, placental, and fetal PR:PROJECT_SUMMARY blood that can have a profound effect on a developing fetus (1, 2). Many PR:PROJECT_SUMMARY metabolites from pregnant mothers cross the placenta to provide energy, PR:PROJECT_SUMMARY structural components, essential nutrients, and signals to the developing fetus PR:PROJECT_SUMMARY (3, 4). Issues with proper transmission of metabolites to the fetus, whether PR:PROJECT_SUMMARY through gestational diabetes, placental insufficiency, or other sources can PR:PROJECT_SUMMARY permanently damage the fetus (5-7). However, quantification of many metabolites PR:PROJECT_SUMMARY entering and exiting the fetus are unknown; associations between microbial PR:PROJECT_SUMMARY metabolites in umbilical cords and disease have not been thoroughly PR:PROJECT_SUMMARY investigated; and there remains a lack of quantifiable metabolic effects of some PR:PROJECT_SUMMARY of the most common medications administered during pregnancy and parturition. PR:PROJECT_SUMMARY Here we identified and quantified many metabolites with a gradient between PR:PROJECT_SUMMARY arterial and venous cord blood; we demonstrated that exogenous metabolites in PR:PROJECT_SUMMARY umbilical cords associate with many health outcomes; and we show that PR:PROJECT_SUMMARY medications can profoundly alter the metabolic milieu of the fetus. We greatly PR:PROJECT_SUMMARY expanded the number of metabolites that demonstrate a gradient between arterial PR:PROJECT_SUMMARY and venous blood, indicating absorption by the fetus, including several PR:PROJECT_SUMMARY essential fatty acids. The microbial metabolites 3-indolepropionic acid, PR:PROJECT_SUMMARY hydroxyhippuric acid and others are associated with many newborn diseases. PR:PROJECT_SUMMARY Lastly, we show that exogenous medications like bupivacaine and betamethasone PR:PROJECT_SUMMARY can have a profound impact on newborn metabolic profile. This study is the most PR:PROJECT_SUMMARY comprehensive study of umbilical cord metabolic and disease associations to PR:PROJECT_SUMMARY date. It reveals important aspects of fetal biology, like the reliance on PR:PROJECT_SUMMARY specific essential fatty acid and taurine. It suggests several interventions in PR:PROJECT_SUMMARY pregnant mothers that may help newborn health, including new fatty acids. This PR:PROJECT_SUMMARY study serves as a valuable reference for investigators wishing to better PR:PROJECT_SUMMARY understand the impact of medications on the developing fetus and neonate. PR:INSTITUTE Stanford University PR:DEPARTMENT Department of Genetics PR:LABORATORY Snyder Lab PR:LAST_NAME Lancaster PR:FIRST_NAME Samuel PR:ADDRESS 240 Pasteur Dr, BMI bldg 4400, Stanford California, 94305 PR:EMAIL slancast@stanford.edu PR:PHONE (612)-600-4033 #STUDY ST:STUDY_TITLE Deep Metabolic Phenotyping of Newborn Cord Blood Reveals Maternal-Fetal ST:STUDY_TITLE Interactions and Disease Risk ST:STUDY_SUMMARY Metabolites are small molecules circulating in the mother, placental, and fetal ST:STUDY_SUMMARY blood that can have a profound effect on a developing fetus (1, 2). Many ST:STUDY_SUMMARY metabolites from pregnant mothers cross the placenta to provide energy, ST:STUDY_SUMMARY structural components, essential nutrients, and signals to the developing fetus ST:STUDY_SUMMARY (3, 4). Issues with proper transmission of metabolites to the fetus, whether ST:STUDY_SUMMARY through gestational diabetes, placental insufficiency, or other sources can ST:STUDY_SUMMARY permanently damage the fetus (5-7). However, quantification of many metabolites ST:STUDY_SUMMARY entering and exiting the fetus are unknown; associations between microbial ST:STUDY_SUMMARY metabolites in umbilical cords and disease have not been thoroughly ST:STUDY_SUMMARY investigated; and there remains a lack of quantifiable metabolic effects of some ST:STUDY_SUMMARY of the most common medications administered during pregnancy and parturition. ST:STUDY_SUMMARY Here we identified and quantified many metabolites with a gradient between ST:STUDY_SUMMARY arterial and venous cord blood; we demonstrated that exogenous metabolites in ST:STUDY_SUMMARY umbilical cords associate with many health outcomes; and we show that ST:STUDY_SUMMARY medications can profoundly alter the metabolic milieu of the fetus. We greatly ST:STUDY_SUMMARY expanded the number of metabolites that demonstrate a gradient between arterial ST:STUDY_SUMMARY and venous blood, indicating absorption by the fetus, including several ST:STUDY_SUMMARY essential fatty acids. The microbial metabolites 3-indolepropionic acid, ST:STUDY_SUMMARY hydroxyhippuric acid and others are associated with many newborn diseases. ST:STUDY_SUMMARY Lastly, we show that exogenous medications like bupivacaine and betamethasone ST:STUDY_SUMMARY can have a profound impact on newborn metabolic profile. This study is the most ST:STUDY_SUMMARY comprehensive study of umbilical cord metabolic and disease associations to ST:STUDY_SUMMARY date. It reveals important aspects of fetal biology, like the reliance on ST:STUDY_SUMMARY specific essential fatty acid and taurine. It suggests several interventions in ST:STUDY_SUMMARY pregnant mothers that may help newborn health, including new fatty acids. This ST:STUDY_SUMMARY study serves as a valuable reference for investigators wishing to better ST:STUDY_SUMMARY understand the impact of medications on the developing fetus and neonate. ST:INSTITUTE Stanford University ST:DEPARTMENT Department of Genetics ST:LABORATORY Snyder Lab ST:LAST_NAME Lancaster ST:FIRST_NAME Samuel ST:ADDRESS 240 Pasteur Dr, BMI bldg 4400, Stanford California, 94305 ST:EMAIL slancast@stanford.edu ST:STUDY_TYPE Untargeted MS and Targeted MS ST:PHONE (612)-600-4033 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 1 type:V RAW_FILE_NAME=1.raw SUBJECT_SAMPLE_FACTORS - 2 type:AV RAW_FILE_NAME=2.raw SUBJECT_SAMPLE_FACTORS - 3 type:NA RAW_FILE_NAME=3.raw SUBJECT_SAMPLE_FACTORS - 4 type:V RAW_FILE_NAME=4.raw SUBJECT_SAMPLE_FACTORS - 5 type:AV RAW_FILE_NAME=5.raw SUBJECT_SAMPLE_FACTORS - 6 type:NA RAW_FILE_NAME=6.raw SUBJECT_SAMPLE_FACTORS - 7 type:AV RAW_FILE_NAME=7.raw SUBJECT_SAMPLE_FACTORS - 7A type:NA RAW_FILE_NAME=7A.raw SUBJECT_SAMPLE_FACTORS - 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CB262-V type:NA RAW_FILE_NAME=CB262-V.raw SUBJECT_SAMPLE_FACTORS - CB263-V type:NA RAW_FILE_NAME=CB263-V.raw SUBJECT_SAMPLE_FACTORS - CB264-A type:NA RAW_FILE_NAME=CB264-A.raw SUBJECT_SAMPLE_FACTORS - CB264-V type:NA RAW_FILE_NAME=CB264-V.raw SUBJECT_SAMPLE_FACTORS - CB265-A type:NA RAW_FILE_NAME=CB265-A.raw SUBJECT_SAMPLE_FACTORS - CB265-V type:NA RAW_FILE_NAME=CB265-V.raw SUBJECT_SAMPLE_FACTORS - CB266-V type:NA RAW_FILE_NAME=CB266-V.raw SUBJECT_SAMPLE_FACTORS - CB267-V type:NA RAW_FILE_NAME=CB267-V.raw SUBJECT_SAMPLE_FACTORS - CB268-V type:NA RAW_FILE_NAME=CB268-V.raw SUBJECT_SAMPLE_FACTORS - CB269-A type:NA RAW_FILE_NAME=CB269-A.raw SUBJECT_SAMPLE_FACTORS - CB269-V type:NA RAW_FILE_NAME=CB269-V.raw #COLLECTION CO:COLLECTION_SUMMARY Plasma was collected from umbilical cords at the Lucile Packard Children’s CO:COLLECTION_SUMMARY Hospital Stanford with permission from the Institutional Review Board (IRB CO:COLLECTION_SUMMARY #46411). All umbilical cords were collected and sent for clinically-indicated CO:COLLECTION_SUMMARY testing to Pathology where blood gas analysis was performed following Stanford CO:COLLECTION_SUMMARY protocol. Leftover and discarded samples were obtained for research. Arterial CO:COLLECTION_SUMMARY and venous blood were extracted from umbilical cords by heparinized syringe and CO:COLLECTION_SUMMARY transferred to low binding tubes. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Samples were centrifuged at 1300g for 10 minutes. Plasma was aliquoted in TR:TREATMENT_SUMMARY cryovials and frozen at -80°C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites and lipids were extracted in 96-well high throughput fashion using a SP:SAMPLEPREP_SUMMARY liquid-liquid biphasic separation with cold methyl tert-butyl ether (MTBE), SP:SAMPLEPREP_SUMMARY methanol, and water. To begin, 1 mL MTBE was added to 40 μl of plasma and SP:SAMPLEPREP_SUMMARY spiked with 40 μl of deuterated lipid internal standards (Sciex, cat# 5040156, SP:SAMPLEPREP_SUMMARY lot# LPISTDKIT-103). The samples were agitated at 4°C for 30 minutes. After the SP:SAMPLEPREP_SUMMARY addition of 250 μl cold water, samples were vortexed for 1 minute then SP:SAMPLEPREP_SUMMARY centrifuged at 3,800 g for 5 minutes at 4°C. The upper organic phase contained SP:SAMPLEPREP_SUMMARY the lipids while the lower aqueous phase contained metabolites with precipitated SP:SAMPLEPREP_SUMMARY proteins at the bottom of the tube. For quality control, reference plasma SP:SAMPLEPREP_SUMMARY samples (40 μl plasma), as well as controls lacking samples (blanks), were SP:SAMPLEPREP_SUMMARY processed in parallel. 1) Metabolites: To further precipitate proteins, 500 μl SP:SAMPLEPREP_SUMMARY 1:1:1 acetone: acetonitrile: methanol spiked with 16 labeled metabolite internal SP:SAMPLEPREP_SUMMARY standards was added to 300 μl of the aqueous phase and 200 μl of the organic SP:SAMPLEPREP_SUMMARY phase and incubated overnight at -20°C. After centrifugation at 3,800 g for 10 SP:SAMPLEPREP_SUMMARY min at 4°C, the metabolic extracts were dried down under a stream of nitrogen SP:SAMPLEPREP_SUMMARY gas and resuspended in 100 μl 50/50 methanol/water for LC-MS. 2) Complex SP:SAMPLEPREP_SUMMARY lipids: 700 µl of the organic phase was dried down under a stream of nitrogen SP:SAMPLEPREP_SUMMARY and resolubilized in 200 μl of methanol for storage at -20°C until analysis. SP:SAMPLEPREP_SUMMARY The day of the analysis, samples were dried down, resuspended in 300 μl of 10 SP:SAMPLEPREP_SUMMARY mM ammonium acetate in 90/10 methanol/toluene, and centrifuged at 3,800 g for 5 SP:SAMPLEPREP_SUMMARY min at 4°C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) CH:SOLVENT_A 10 mM ammonium acetate in 50/50 acetonitrile/water CH:SOLVENT_B 10 mM ammonium acetate in 95/5 acetonitrile/water CH:FLOW_GRADIENT N/A CH:FLOW_RATE 0.5 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data from each mode were independently analyzed using Progenesis QI software MS:MS_COMMENTS (v2.3) (Nonlinear Dynamics, Durham, NC). Metabolic features from blanks and MS:MS_COMMENTS those that did not show sufficient linearity upon dilution in QC samples (r<0.6) MS:MS_COMMENTS were discarded. Only metabolic features present in >2/3 of the samples were kept MS:MS_COMMENTS for further analysis. Missing values were imputed by drawing from a random MS:MS_COMMENTS distribution of low values in the corresponding sample. Intensity drift was MS:MS_COMMENTS corrected using SERRF. Data quality post-normalization was verified by ensuring MS:MS_COMMENTS clustering of pooled sample replicates on a principal component analysis (PCA) MS:MS_COMMENTS plot. MS:MS_RESULTS_FILE ST002937_AN004820_Results.txt UNITS:Relative Abundance Has m/z:Yes Has RT:Yes RT units:Minutes #END