Summary of study ST000092

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 PR000084. The data can be accessed directly via it's Project DOI: 10.21228/M8PK5V This work is supported by NIH grant, U2C- DK119886.

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Study IDST000092
Study TitleA statistical analysis of the effects of urease pre-treatment on the measurement of the urinary metabolome by gas chromatography–mass spectrometry
Study TypeAnalytical Comparison
Study SummaryUrease pre-treatment of urine has been utilized since the early 1960s to remove high levels of urea from samples prior to further processing and analysis by gas chromatography–mass spectrometry (GC–MS). Aside from the obvious depletion or elimination of urea, the effect, if any, of urease pre-treatment on the urinary metabolome has not been studied in detail. Here, we report the results of three separate but related experiments that were designed to assess possible indirect effects of urease pre-treatment on the urinary metabolome as measured by GC–MS. In total, 235 GC–MS analyses were performed and over 106 identified and 200 unidentified metabolites were quantified across the three experiments. The results showed that data from urease pre-treated samples (1) had the same or lower coefficients of variance among reproducibly detected metabolites, (2) more accurately reflected quantitative differences and the expected ratios among different urine volumes, and (3) increased the number of metabolite identifications. Overall, we observed no negative consequences of urease pre-treatment. In contrast, urease pre-treatment enhanced the ability to distinguish between volume-based and biological sample types compared to no treatment. Taken together, these results show that urease pre-treatment of urine offers multiple beneficial effects that outweigh any artifacts that may be introduced to the data in urinary metabolomics analyses.
Institute
Pacific Northwest National Laboratory
DepartmentBiological Separation and Mass Spectrometry
Last NameMetz
First NameThomas
Emailthomas.metz@pnnl.gov
Submit Date2014-06-25
Num Groups6
Total Subjects235
Raw Data AvailableYes
Raw Data File Type(s).cdf
Uploaded File Size3.0 G
Analysis Type DetailGC-MS
Release Date2014-08-07
Release Version1
Thomas Metz Thomas Metz
https://dx.doi.org/10.21228/M8PK5V
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000084
Project DOI:doi: 10.21228/M8PK5V
Project Title:T1D Investigating the gut microbiome, urinary proteome, and metabolome
Project Type:MS analysis
Institute:J. Craig Venter Institute
Department:Genomic Medicine Group
Last Name:Madupu
First Name:Ramana
Email:rmadupu@jcvi.org

Subject:

Subject ID:SU000111
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Male and Female
Human Race:Hispanic, Aisan, Caucasian
Species Group:Human

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Urease Treatment Volume Urine (µL) Gender Solution Added Ethnicity
SA005020VaryingVolume_NT_100uL_1No treatment 100 N/A N/A N/A
SA005021VaryingVolume_NT_100uL_3No treatment 100 N/A N/A N/A
SA005022VaryingVolume_NT_100uL_2No treatment 100 N/A N/A N/A
SA005023ConstantVolume_NT_4_3No treatment 100 N/A None N/A
SA005024ConstantVolume_NT_4_2No treatment 100 N/A None N/A
SA005025ConstantVolume_NT_5_1No treatment 100 N/A None N/A
SA005026ConstantVolume_NT_5_3No treatment 100 N/A None N/A
SA005027ConstantVolume_NT_1_1No treatment 100 N/A None N/A
SA005028ConstantVolume_NT_4_1No treatment 100 N/A None N/A
SA005029ConstantVolume_NT_5_2No treatment 100 N/A None N/A
SA005030ConstantVolume_NT_2_1No treatment 100 N/A None N/A
SA005031ConstantVolume_NT_1_3No treatment 100 N/A None N/A
SA005032ConstantVolume_NT_3_3No treatment 100 N/A None N/A
SA005033ConstantVolume_NT_1_2No treatment 100 N/A None N/A
SA005034ConstantVolume_NT_2_2No treatment 100 N/A None N/A
SA005035ConstantVolume_NT_3_2No treatment 100 N/A None N/A
SA005036ConstantVolume_NT_3_1No treatment 100 N/A None N/A
SA005037ConstantVolume_NT_2_3No treatment 100 N/A None N/A
SA005038VaryingVolume_NT_10uL_3No treatment 10 N/A N/A N/A
SA005039VaryingVolume_NT_10uL_1No treatment 10 N/A N/A N/A
SA005040VaryingVolume_NT_10uL_2No treatment 10 N/A N/A N/A
SA005041VaryingVolume_NT_25uL_2No treatment 25 N/A N/A N/A
SA005042VaryingVolume_NT_25uL_3No treatment 25 N/A N/A N/A
SA005043VaryingVolume_NT_25uL_1No treatment 25 N/A N/A N/A
SA005044MaleVsFemale_NT_Female_Asian_24_BRH600219_DNo treatment 50 Female N/A Asian
SA005045MaleVsFemale_NT_Female_Asian_23_BRH600227_BNo treatment 50 Female N/A Asian
SA005046MaleVsFemale_NT_Female_Asian_24_BRH600219_CNo treatment 50 Female N/A Asian
SA005047MaleVsFemale_NT_Female_Asian_23_BRH600227_ANo treatment 50 Female N/A Asian
SA005048MaleVsFemale_NT_Female_Black_24_BRH600216_BNo treatment 50 Female N/A Black
SA005049MaleVsFemale_NT_Female_Black_24_BRH600226_CNo treatment 50 Female N/A Black
SA005050MaleVsFemale_NT_Female_Black_24_BRH600216_ANo treatment 50 Female N/A Black
SA005051MaleVsFemale_NT_Female_Black_24_BRH600226_DNo treatment 50 Female N/A Black
SA005052MaleVsFemale_NT_Female_Caucasian_24_BRH600211_ANo treatment 50 Female N/A Caucasian
SA005053MaleVsFemale_NT_Female_Caucasian_24_BRH600211_BNo treatment 50 Female N/A Caucasian
SA005054MaleVsFemale_NT_Female_Caucasian_23_BRH600228_BNo treatment 50 Female N/A Caucasian
SA005055MaleVsFemale_NT_Female_Caucasian_23_BRH600228_ANo treatment 50 Female N/A Caucasian
SA005056MaleVsFemale_NT_Female_Caucasian_23_BRH600225_CNo treatment 50 Female N/A Caucasian
SA005057MaleVsFemale_NT_Female_Caucasian_23_BRH600225_DNo treatment 50 Female N/A Caucasian
SA005058MaleVsFemale_NT_Female_Caucasian_24_BRH600215_CNo treatment 50 Female N/A Caucasian
SA005059MaleVsFemale_NT_Female_Caucasian_24_BRH600215_DNo treatment 50 Female N/A Caucasian
SA005060MaleVsFemale_NT_Female_Caucasian_24_BRH600223_BNo treatment 50 Female N/A Caucasian
SA005061MaleVsFemale_NT_Female_Caucasian_24_BRH600217_DNo treatment 50 Female N/A Caucasian
SA005062MaleVsFemale_NT_Female_Caucasian_24_BRH600229_ANo treatment 50 Female N/A Caucasian
SA005063MaleVsFemale_NT_Female_Caucasian_24_BRH600229_BNo treatment 50 Female N/A Caucasian
SA005064MaleVsFemale_NT_Female_Caucasian_24_BRH600217_CNo treatment 50 Female N/A Caucasian
SA005065MaleVsFemale_NT_Female_Caucasian_23_BRH600224_DNo treatment 50 Female N/A Caucasian
SA005066MaleVsFemale_NT_Female_Caucasian_24_BRH600223_ANo treatment 50 Female N/A Caucasian
SA005067MaleVsFemale_NT_Female_Caucasian_23_BRH600218_ANo treatment 50 Female N/A Caucasian
SA005068MaleVsFemale_NT_Female_Caucasian_23_BRH600224_CNo treatment 50 Female N/A Caucasian
SA005069MaleVsFemale_NT_Female_Caucasian_22_BRH600221_DNo treatment 50 Female N/A Caucasian
SA005070MaleVsFemale_NT_Female_Caucasian_22_BRH600221_CNo treatment 50 Female N/A Caucasian
SA005071MaleVsFemale_NT_Female_Caucasian_23_BRH600213_DNo treatment 50 Female N/A Caucasian
SA005072MaleVsFemale_NT_Female_Caucasian_23_BRH600213_CNo treatment 50 Female N/A Caucasian
SA005073MaleVsFemale_NT_Female_Caucasian_23_BRH600218_BNo treatment 50 Female N/A Caucasian
SA005074MaleVsFemale_NT_Female_Caucasian_23_BRH600214_BNo treatment 50 Female N/A Caucasian
SA005075MaleVsFemale_NT_Female_Caucasian_23_BRH600214_ANo treatment 50 Female N/A Caucasian
SA005076MaleVsFemale_NT_Female_Hispanic_23_BRH600210_BNo treatment 50 Female N/A Hispanic
SA005077MaleVsFemale_NT_Female_Hispanic_23_BRH600212_DNo treatment 50 Female N/A Hispanic
SA005078MaleVsFemale_NT_Female_Hispanic_24_BRH600220_ANo treatment 50 Female N/A Hispanic
SA005079MaleVsFemale_NT_Female_Hispanic_24_BRH600222_DNo treatment 50 Female N/A Hispanic
SA005080MaleVsFemale_NT_Female_Hispanic_23_BRH600212_CNo treatment 50 Female N/A Hispanic
SA005081MaleVsFemale_NT_Female_Hispanic_24_BRH600220_BNo treatment 50 Female N/A Hispanic
SA005082MaleVsFemale_NT_Female_Hispanic_24_BRH600222_CNo treatment 50 Female N/A Hispanic
SA005083MaleVsFemale_NT_Female_Hispanic_23_BRH600210_ANo treatment 50 Female N/A Hispanic
SA005084MaleVsFemale_NT_Male_Caucasian_21_BRH600204_DNo treatment 50 Male N/A Caucasian
SA005085MaleVsFemale_NT_Male_Caucasian_22_BRH600190_ANo treatment 50 Male N/A Caucasian
SA005086MaleVsFemale_NT_Male_Caucasian_21_BRH600204_CNo treatment 50 Male N/A Caucasian
SA005087MaleVsFemale_NT_Male_Caucasian_21_BRH600202_DNo treatment 50 Male N/A Caucasian
SA005088MaleVsFemale_NT_Male_Caucasian_21_BRH600202_CNo treatment 50 Male N/A Caucasian
SA005089MaleVsFemale_NT_Male_Caucasian_22_BRH600190_BNo treatment 50 Male N/A Caucasian
SA005090MaleVsFemale_NT_Male_Caucasian_22_BRH600194_DNo treatment 50 Male N/A Caucasian
SA005091MaleVsFemale_NT_Male_Caucasian_22_BRH600199_CNo treatment 50 Male N/A Caucasian
SA005092MaleVsFemale_NT_Male_Caucasian_22_BRH600199_DNo treatment 50 Male N/A Caucasian
SA005093MaleVsFemale_NT_Male_Caucasian_22_BRH600198_BNo treatment 50 Male N/A Caucasian
SA005094MaleVsFemale_NT_Male_Caucasian_22_BRH600198_ANo treatment 50 Male N/A Caucasian
SA005095MaleVsFemale_NT_Male_Caucasian_21_BRH600201_ANo treatment 50 Male N/A Caucasian
SA005096MaleVsFemale_NT_Male_Caucasian_22_BRH600194_CNo treatment 50 Male N/A Caucasian
SA005097MaleVsFemale_NT_Male_Caucasian_21_BRH600201_BNo treatment 50 Male N/A Caucasian
SA005098MaleVsFemale_NT_Male_Caucasian_20_BRH600193_BNo treatment 50 Male N/A Caucasian
SA005099MaleVsFemale_NT_Male_Caucasian_20_BRH600193_ANo treatment 50 Male N/A Caucasian
SA005100MaleVsFemale_NT_Male_Caucasian_21_BRH600197_DNo treatment 50 Male N/A Caucasian
SA005101MaleVsFemale_NT_Male_Caucasian_20_BRH600196_BNo treatment 50 Male N/A Caucasian
SA005102MaleVsFemale_NT_Male_Caucasian_20_BRH600196_ANo treatment 50 Male N/A Caucasian
SA005103MaleVsFemale_NT_Male_Caucasian_21_BRH600192_ANo treatment 50 Male N/A Caucasian
SA005104MaleVsFemale_NT_Male_Caucasian_21_BRH600192_BNo treatment 50 Male N/A Caucasian
SA005105MaleVsFemale_NT_Male_Caucasian_21_BRH600197_CNo treatment 50 Male N/A Caucasian
SA005106MaleVsFemale_NT_Male_Hispanic_21_BRH600209_CNo treatment 50 Male N/A Hispanic
SA005107MaleVsFemale_NT_Male_Hispanic_21_BRH600205_BNo treatment 50 Male N/A Hispanic
SA005108MaleVsFemale_NT_Male_Hispanic_22_BRH600203_BNo treatment 50 Male N/A Hispanic
SA005109MaleVsFemale_NT_Male_Hispanic_21_BRH600191_BNo treatment 50 Male N/A Hispanic
SA005110MaleVsFemale_NT_Male_Hispanic_21_BRH600209_DNo treatment 50 Male N/A Hispanic
SA005111MaleVsFemale_NT_Male_Hispanic_22_BRH600195_CNo treatment 50 Male N/A Hispanic
SA005112MaleVsFemale_NT_Male_Hispanic_21_BRH600191_ANo treatment 50 Male N/A Hispanic
SA005113MaleVsFemale_NT_Male_Hispanic_22_BRH600195_DNo treatment 50 Male N/A Hispanic
SA005114MaleVsFemale_NT_Male_Hispanic_22_BRH600200_ANo treatment 50 Male N/A Hispanic
SA005115MaleVsFemale_NT_Male_Hispanic_22_BRH600200_BNo treatment 50 Male N/A Hispanic
SA005116MaleVsFemale_NT_Male_Hispanic_22_BRH600203_ANo treatment 50 Male N/A Hispanic
SA005117MaleVsFemale_NT_Male_Hispanic_21_BRH600205_ANo treatment 50 Male N/A Hispanic
SA005118MaleVsFemale_NT_Male_Hispanic_20_BRH600206_CNo treatment 50 Male N/A Hispanic
SA005119MaleVsFemale_NT_Male_Hispanic_20_BRH600208_DNo treatment 50 Male N/A Hispanic
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Collection:

Collection ID:CO000094
Collection Summary:Approval for the conduct of this programmatic research was obtained from the Pacific Northwest National Laboratory Institutional Review Board. Urine samples from consenting male and female donors (n = 20 each, Supplemental Table S1) after an overnight fast were purchased from Bioreclamation, LLC (Hicksville, NY) and received frozen on dry ice and deidentified. To create a uniform sample for Experiments 1 and 2 (see below), aliquots from each individual sample were pooled, realiquoted, and stored at -80C until used.
Sample Type:Urine
Storage Conditions:-80° C

Treatment:

Treatment ID:TR000112
Treatment Summary:Varying Volume with Urease / Male vs. Female with Urease / Constant Volume with Urease / Constant Volume with 100 µL Water
Treatment Protocol Comments:To evaluate whether the effects of urease pre-treatment of urine varied with the volume of urine prepared, we compared the urine metabolite profiles from pooled urine after pre-treatment with urease (UT) and after no treatment (NT) using various volumes of urine. For this, several volumes (5, 10, 25, 50, and 100 µL) of the pooled urine sample were incubated (n = 3, each) with 100 µL of a 1 mg/mL solution of urease or were not subjected to any treatment. / Finally, to evaluate whether any artifacts introduced by urease pre-treatment on the urinary metabolome interfered with the ability to distinguish between comparative samples, we compared the metabolite profiles from individual male and female urine samples after pre-treatment with urease or after no treatment (previous metabolomics studies of male and female urines (Pasikanti et al., 2008; Slupsky et al., 2007; Saude et al., 2007; Psihogios et al., 2008) have reported differences in metabolite levels). For this, 50 µL aliquots of individual male and female urine samples (n = 20, each) were blocked, randomized, and then incubated with 50 µL of a 1 mg/mL solution of urease (UT) or were not subjected to any treatment (NT), each as described above. / To initially evaluate the effects of urease pre-treatment on the urinary metabolome, we compared the urine metabolite profiles from pooled urine after pre-treatment with urease, water, or no treatment at all. For this, 100 uL aliquots of the pooled urine sample were incubated (n = 5, each) with 100 uL of a 1 mg/mL solution of urease (Sigma-Aldrich catalog number U4002) prepared in water (urease-treated; ‘UT’) or an equal volume of water alone (water-treated; ‘WT’) for 30 min at 37 C with mild shaking (500 rpm). Identical aliquots (n = 5) were not subjected to any treatment (no treatment; ‘NT’) and allowed to sit at room temperature for 30 min. / To initially evaluate the effects of urease pre-treatment on the urinary metabolome, we compared the urine metabolite profiles from pooled urine after pre-treatment with urease, water, or no treatment at all. For this, 100 uL aliquots of the pooled urine sample were incubated (n = 5, each) with 100 uL of a 1 mg/mL solution of urease (Sigma-Aldrich catalog number U4002) prepared in water (urease-treated; ‘UT’) or an equal volume of water alone (water-treated; ‘WT’) for 30 min at 37 C with mild shaking (500 rpm). Identical aliquots (n = 5) were not subjected to any treatment (no treatment; ‘NT’) and allowed to sit at room temperature for 30 min.
Treatment:Abiotic
Treatment Compound:Urease / Urease / Urease / Water
Treatment Dose:1 mg/ml / 1 mg/ml / 1 mg/ml / --
Treatment Dosevolume:100 µL / 50 µL / 100 µL / 100 µL
Human Fasting:Overnight Fast

Sample Preparation:

Sampleprep ID:SP000107
Sampleprep Summary:concomitant protein precipitation with cold methanol, vortexing, centrifugation, supernatent dried in vacuo, stored at -80° c, chemical derivitization
Sampleprep Protocol Comments:Metabolites were extracted with concomitant protein precipitation by addition of 1 mL of cold (-20 C) methanol with vortexing for 30 s, and precipitated proteins were removed by centrifugation at 15,000xg for 10 min at 4 C. The supernatants were transferred to glass autosampler vials and then dried in vacuo prior to chemical derivatization. If the extracts could not be immediately derivatized an d analyzed by GC–MS, then they were stored at -80C. Dried metabolite extracts were chemically derivatized using a modified version of the protocol used to create FiehnLib (Kind et al., 2009). Briefly, dried metabolite extracts were dried again to remove any residual water if they had been stored at -80°C. To protect carbonyl groups and reduce the number of tautomeric isomers, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by vortexing for 30 s and incubation at 37°C with generous shaking (1000 rpm) for 90 min. At this point, the sample vials were inverted one time to capture any condensation of solvent at the cap surface, followed by a brief centrifugation at 1000×g for 1 min. To derivatize hydroxyl and amine groups to trimethylsilyated (TMS) forms, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were then added to each vial, followed by vortexing for 10 s and incubation at 37°C with shaking (1000 rpm) for 30 min. Again, the sample vials were inverted one time, followed by centrifugation at 1000×g for 5 min. The samples were allowed to cool to room temperature and were analyzed in the same day.
Processing Method:precipitation, centrifugation
Processing Storage Conditions:dried in vacuo, stored at -80° C
Extraction Method:concomitant protein precipitation by addition of 1 mL of cold (-20 C) methanol with vortexing and centrifugation
Extract Enrichment:dried in vacuo
Extract Storage:-80° C
Sample Resuspension:20 µL of methoxyamine in pyridine (30 mg/mL)
Sample Derivatization:20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS)

Combined analysis:

Analysis ID AN000146
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890A
Column Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5975C
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH000104
Chromatography Summary:Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation
Chromatography Comments:Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min.
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Flow Rate:0.450.5 mL/min
Injection Temperature:250 C
Sample Injection:1 L, splitless
Analytical Time:37.5 min
Oven Temperature:60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C
Sample Syringe Size:10 L
Chromatography Type:GC

MS:

MS ID:MS000122
Analysis ID:AN000146
Instrument Name:Agilent 5975C
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis.
Ion Mode:POSITIVE
Scan Range Moverz:50-550 m/z
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