#METABOLOMICS WORKBENCH ReemAlMalki91_20230328_085203 DATATRACK_ID:3823 STUDY_ID:ST002552 ANALYSIS_ID:AN004202 PROJECT_ID:PR001644 VERSION 1 CREATED_ON April 6, 2023, 3:03 pm #PROJECT PR:PROJECT_TITLE Biomarker discovery in galactosemia: Metabolomics with UPLC/HRMS in dried blood PR:PROJECT_TITLE spots PR:PROJECT_TYPE newborn screening PR:PROJECT_SUMMARY Galactosemia (GAL) is an autosomal recessive genetic disorder characterized by PR:PROJECT_SUMMARY galactose metabolism disturbances. GAL develops non-preventable life-threatening PR:PROJECT_SUMMARY complications even with a reduced content of galactose and lactose patient’s PR:PROJECT_SUMMARY diet. Thus, the underlying pathophysiology of long-term complications in GAL PR:PROJECT_SUMMARY remains poorly understood. The current study used a metabolomics approach using PR:PROJECT_SUMMARY ultra-performance liquid chromatography coupled with high-resolution mass PR:PROJECT_SUMMARY spectrometry to investigate the metabolomic changes in the dried blood spots of PR:PROJECT_SUMMARY 15 patients with GAL and 39 healthy individuals. Compared to the control group, PR:PROJECT_SUMMARY 2,819 metabolites underwent significant changes in patients with GAL. In all, PR:PROJECT_SUMMARY 480 human endogenous metabolites were identified, of which 209 and 271 were PR:PROJECT_SUMMARY upregulated and downregulated, respectively. PA (8:0/LTE4) and ganglioside GT1c PR:PROJECT_SUMMARY (d18:0/20:0) metabolites showed the most significant difference between GAL and PR:PROJECT_SUMMARY the healthy group, with an area under the curve of 1 and 0.995, respectively. PR:PROJECT_SUMMARY Additionally, our findings showed novel potential biomarkers for GAL, such as PR:PROJECT_SUMMARY 17-alpha-estradiol-3-glucuronide and 16-alpha-hydroxy DHEA 3-sulfatediphosphate. PR:PROJECT_SUMMARY In conclusion, this metabolomics study deepened the understanding of the PR:PROJECT_SUMMARY pathophysiology of GAL and presented metabolites that might serve as potential PR:PROJECT_SUMMARY prognostic biomarkers to monitor the progression or support the clinical PR:PROJECT_SUMMARY diagnosis of GAL. PR:INSTITUTE King Saud University PR:DEPARTMENT Metabolomics PR:LABORATORY Metabolomics PR:LAST_NAME AlMalki PR:FIRST_NAME Reem PR:ADDRESS King Fahad road, Riyadh, KSA, 00000, Saudi Arabia PR:EMAIL 439203044@student.ksu.edu.sa PR:PHONE +966534045397 #STUDY ST:STUDY_TITLE Biomarker discovery in galactosemia: Metabolomics with UPLC/HRMS in dried blood ST:STUDY_TITLE spots ST:STUDY_TYPE Newborn screening ST:STUDY_SUMMARY Galactosemia (GAL) is an autosomal recessive genetic disorder characterized by ST:STUDY_SUMMARY galactose metabolism disturbances. GAL develops non-preventable life-threatening ST:STUDY_SUMMARY complications even with a reduced content of galactose and lactose patient’s ST:STUDY_SUMMARY diet. Thus, the underlying pathophysiology of long-term complications in GAL ST:STUDY_SUMMARY remains poorly understood. The current study used a metabolomics approach using ST:STUDY_SUMMARY ultra-performance liquid chromatography coupled with high-resolution mass ST:STUDY_SUMMARY spectrometry to investigate the metabolomic changes in the dried blood spots of ST:STUDY_SUMMARY 15 patients with GAL and 39 healthy individuals. Compared to the control group, ST:STUDY_SUMMARY 2,819 metabolites underwent significant changes in patients with GAL. In all, ST:STUDY_SUMMARY 480 human endogenous metabolites were identified, of which 209 and 271 were ST:STUDY_SUMMARY upregulated and downregulated, respectively. PA (8:0/LTE4) and ganglioside GT1c ST:STUDY_SUMMARY (d18:0/20:0) metabolites showed the most significant difference between GAL and ST:STUDY_SUMMARY the healthy group, with an area under the curve of 1 and 0.995, respectively. ST:STUDY_SUMMARY Additionally, our findings showed novel potential biomarkers for GAL, such as ST:STUDY_SUMMARY 17-alpha-estradiol-3-glucuronide and 16-alpha-hydroxy DHEA 3-sulfatediphosphate. ST:STUDY_SUMMARY In conclusion, this metabolomics study deepened the understanding of the ST:STUDY_SUMMARY pathophysiology of GAL and presented metabolites that might serve as potential ST:STUDY_SUMMARY prognostic biomarkers to monitor the progression or support the clinical ST:STUDY_SUMMARY diagnosis of GAL. ST:INSTITUTE King Saud University ST:LAST_NAME AlMalki ST:FIRST_NAME Reem ST:ADDRESS King Fahad road, Riyadh, KSA, 00000, Saudi Arabia ST:EMAIL 439203044@student.ksu.edu.sa ST:PHONE +966534045397 ST:NUM_GROUPS 2 ST:PUBLICATIONS Yes #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #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 Patient GALT_AB1_20587853 Factor:Patient RAW_FILE_NAME=GALT_AB1_20587853.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB2_21780341 Factor:Patient RAW_FILE_NAME=GALT_AB2_21780341.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB3_21745506 Factor:Patient RAW_FILE_NAME=GALT_AB3_21745506.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB4_21100457 Factor:Patient RAW_FILE_NAME=GALT_AB4_21100457.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB6_21770900 Factor:Patient RAW_FILE_NAME=GALT_AB6_21770900.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB8_21112212 Factor:Patient RAW_FILE_NAME=GALT_AB8_21112212.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB9_21775172 Factor:Patient RAW_FILE_NAME=GALT_AB9_21775172.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB12_21780943 Factor:Patient RAW_FILE_NAME=GALT_AB12_21780943.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB13_21769782 Factor:Patient RAW_FILE_NAME=GALT_AB13_21769782.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB14_21799507 Factor:Patient RAW_FILE_NAME=GALT_AB14_21799507.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB15_21745524 Factor:Patient RAW_FILE_NAME=GALT_AB15_21745524.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB16_20428037 Factor:Patient RAW_FILE_NAME=GALT_AB16_20428037.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB17_20758512 Factor:Patient RAW_FILE_NAME=GALT_AB17_20758512.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB18_21012310 Factor:Patient RAW_FILE_NAME=GALT_AB18_21012310.raw SUBJECT_SAMPLE_FACTORS Patient GALT_AB19_21744172 Factor:Patient RAW_FILE_NAME=GALT_AB19_21744172.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR1_21799701 Factor:Ctrl RAW_FILE_NAME=GALT_NR1_21799701.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR2_21799835 Factor:Ctrl RAW_FILE_NAME=GALT_NR2_21799835.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR3_21799729 Factor:Ctrl RAW_FILE_NAME=GALT_NR3_21799729.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR4_21752056 Factor:Ctrl RAW_FILE_NAME=GALT_NR4_21752056.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR5_21756742 Factor:Ctrl RAW_FILE_NAME=GALT_NR5_21756742.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR6_21799686 Factor:Ctrl RAW_FILE_NAME=GALT_NR6_21799686.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR7_21798410 Factor:Ctrl RAW_FILE_NAME=GALT_NR7_21798410.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR8_21756982 Factor:Ctrl RAW_FILE_NAME=GALT_NR8_21756982.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR9_21756292 Factor:Ctrl RAW_FILE_NAME=GALT_NR9_21756292.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR10_21756779 Factor:Ctrl RAW_FILE_NAME=GALT_NR10_21756779.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR11_21798863 Factor:Ctrl RAW_FILE_NAME=GALT_NR11_21798863.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR12_21798942 Factor:Ctrl RAW_FILE_NAME=GALT_NR12_21798942.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR13_21756636 Factor:Ctrl RAW_FILE_NAME=GALT_NR13_21756636.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR14_21756609 Factor:Ctrl RAW_FILE_NAME=GALT_NR14_21756609.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR15_21798906 Factor:Ctrl RAW_FILE_NAME=GALT_NR15_21798906.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR16_21794987 Factor:Ctrl RAW_FILE_NAME=GALT_NR16_21794987.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR17_21798924 Factor:Ctrl RAW_FILE_NAME=GALT_NR17_21798924.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR18_21756973 Factor:Ctrl RAW_FILE_NAME=GALT_NR18_21756973.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR19_21756645 Factor:Ctrl RAW_FILE_NAME=GALT_NR19_21756645.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR20_21798933 Factor:Ctrl RAW_FILE_NAME=GALT_NR20_21798933.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR21_21756964 Factor:Ctrl RAW_FILE_NAME=GALT_NR21_21756964.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR22_21799695 Factor:Ctrl RAW_FILE_NAME=GALT_NR22_21799695.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR23_21752092 Factor:Ctrl RAW_FILE_NAME=GALT_NR23_21752092.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR24_21799792 Factor:Ctrl RAW_FILE_NAME=GALT_NR24_21799792.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR25_21757006 Factor:Ctrl RAW_FILE_NAME=GALT_NR25_21757006.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR26_21799932 Factor:Ctrl RAW_FILE_NAME=GALT_NR26_21799932.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR28_21799871 Factor:Ctrl RAW_FILE_NAME=GALT_NR28_21799871.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR29_21798155 Factor:Ctrl RAW_FILE_NAME=GALT_NR29_21798155.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR30_21799710 Factor:Ctrl RAW_FILE_NAME=GALT_NR30_21799710.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR31_21756654 Factor:Ctrl RAW_FILE_NAME=GALT_NR31_21756654.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR32_21799969 Factor:Ctrl RAW_FILE_NAME=GALT_NR32_21799969.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR33_21798474 Factor:Ctrl RAW_FILE_NAME=GALT_NR33_21798474.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR34_21799978 Factor:Ctrl RAW_FILE_NAME=GALT_NR34_21799978.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR35_21799853 Factor:Ctrl RAW_FILE_NAME=GALT_NR35_21799853.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR36_21799941 Factor:Ctrl RAW_FILE_NAME=GALT_NR36_21799941.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR37_21798960 Factor:Ctrl RAW_FILE_NAME=GALT_NR37_21798960.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR38_21783126 Factor:Ctrl RAW_FILE_NAME=GALT_NR38_21783126.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR39_21799677 Factor:Ctrl RAW_FILE_NAME=GALT_NR39_21799677.raw SUBJECT_SAMPLE_FACTORS Ctrl GALT_NR40_21798479 Factor:Ctrl RAW_FILE_NAME=GALT_NR40_21798479.raw #COLLECTION CO:COLLECTION_SUMMARY Fifty-four DBS samples were collected from genetically and biochemically CO:COLLECTION_SUMMARY confirmed GAL (n = 15) patients at King Faisal Specialist Hospital and Research CO:COLLECTION_SUMMARY center (KFSHRC) and healthy controls (n = 39). CO:COLLECTION_PROTOCOL_FILENAME Characteristics of the study population and metabolites extraction CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY no treatment use #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites extraction The polar metabolites were extracted from DBS samples SP:SAMPLEPREP_SUMMARY using our developed standard protocol (Jacob et al., 2018). Five 3 mm size DBS SP:SAMPLEPREP_SUMMARY disks were used for metabolite extraction using methanol, acetonitrile, and SP:SAMPLEPREP_SUMMARY water (40:40:20%) for protein precipitation. The mixture was mixed at 25°C and SP:SAMPLEPREP_SUMMARY 600 rpm for 2 hours in a thermomixer (Eppendorf, Germany). Pooled QC samples SP:SAMPLEPREP_SUMMARY were prepared using aliquots from the study samples. Afterward, the supernatants SP:SAMPLEPREP_SUMMARY were transferred to another set of tubes, evaporated in SpeedVacc (Christ, City, SP:SAMPLEPREP_SUMMARY Germany), and stored at −80°C until LCMS analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME Metabolites extraction SP:PROCESSING_STORAGE_CONDITIONS -20℃ SP:EXTRACT_STORAGE Room temperature #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity UPLC XSelect HSS C18 (100 × 2.1mm, 2.5um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 50% methanol/50% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0–16 min 95%–5% A, 16–19 min 5% A, 19–20 min 5%–95% A, and 20–22 CH:FLOW_GRADIENT min, 95%– 95% A CH:FLOW_RATE 300 μl/min. CH:COLUMN_TEMPERATURE 55 CH:METHODS_FILENAME UPLCHRMS #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The DIA data were collected with a Masslynx™ V4.1 workstation in continuum MS:MS_COMMENTS mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a MS:MS_COMMENTS standard pipeline using the Progenesis QI v.3.0 software. MS:MS_RESULTS_FILE ST002552_AN004202_Results.txt UNITS:peak area Has m/z:Yes Has RT:No RT units:Minutes #END