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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST000092
Study Title	A statistical analysis of the effects of urease pre-treatment on the measurement of the urinary metabolome by gas chromatographymass spectrometry
Study Type	Analytical Comparison
Study Summary	Urease 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 chromatographymass spectrometry (GCMS). 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 GCMS. In total, 235 GCMS 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
Department	Biological Separation and Mass Spectrometry
Last Name	Metz
First Name	Thomas
Email	thomas.metz@pnnl.gov
Submit Date	2014-06-25
Num Groups	6
Total Subjects	235
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Uploaded File Size	3.0 G
Analysis Type Detail	GC-MS
Release Date	2014-08-07
Release Version	1

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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
SA005020	VaryingVolume_NT_100uL_1	No treatment	100	N/A	N/A	N/A
SA005021	VaryingVolume_NT_100uL_3	No treatment	100	N/A	N/A	N/A
SA005022	VaryingVolume_NT_100uL_2	No treatment	100	N/A	N/A	N/A
SA005023	ConstantVolume_NT_4_3	No treatment	100	N/A	None	N/A
SA005024	ConstantVolume_NT_4_2	No treatment	100	N/A	None	N/A
SA005025	ConstantVolume_NT_5_1	No treatment	100	N/A	None	N/A
SA005026	ConstantVolume_NT_5_3	No treatment	100	N/A	None	N/A
SA005027	ConstantVolume_NT_1_1	No treatment	100	N/A	None	N/A
SA005028	ConstantVolume_NT_4_1	No treatment	100	N/A	None	N/A
SA005029	ConstantVolume_NT_5_2	No treatment	100	N/A	None	N/A
SA005030	ConstantVolume_NT_2_1	No treatment	100	N/A	None	N/A
SA005031	ConstantVolume_NT_1_3	No treatment	100	N/A	None	N/A
SA005032	ConstantVolume_NT_3_3	No treatment	100	N/A	None	N/A
SA005033	ConstantVolume_NT_1_2	No treatment	100	N/A	None	N/A
SA005034	ConstantVolume_NT_2_2	No treatment	100	N/A	None	N/A
SA005035	ConstantVolume_NT_3_2	No treatment	100	N/A	None	N/A
SA005036	ConstantVolume_NT_3_1	No treatment	100	N/A	None	N/A
SA005037	ConstantVolume_NT_2_3	No treatment	100	N/A	None	N/A
SA005038	VaryingVolume_NT_10uL_3	No treatment	10	N/A	N/A	N/A
SA005039	VaryingVolume_NT_10uL_1	No treatment	10	N/A	N/A	N/A
SA005040	VaryingVolume_NT_10uL_2	No treatment	10	N/A	N/A	N/A
SA005041	VaryingVolume_NT_25uL_2	No treatment	25	N/A	N/A	N/A
SA005042	VaryingVolume_NT_25uL_3	No treatment	25	N/A	N/A	N/A
SA005043	VaryingVolume_NT_25uL_1	No treatment	25	N/A	N/A	N/A
SA005044	MaleVsFemale_NT_Female_Asian_24_BRH600219_D	No treatment	50	Female	N/A	Asian
SA005045	MaleVsFemale_NT_Female_Asian_23_BRH600227_B	No treatment	50	Female	N/A	Asian
SA005046	MaleVsFemale_NT_Female_Asian_24_BRH600219_C	No treatment	50	Female	N/A	Asian
SA005047	MaleVsFemale_NT_Female_Asian_23_BRH600227_A	No treatment	50	Female	N/A	Asian
SA005048	MaleVsFemale_NT_Female_Black_24_BRH600216_B	No treatment	50	Female	N/A	Black
SA005049	MaleVsFemale_NT_Female_Black_24_BRH600226_C	No treatment	50	Female	N/A	Black
SA005050	MaleVsFemale_NT_Female_Black_24_BRH600216_A	No treatment	50	Female	N/A	Black
SA005051	MaleVsFemale_NT_Female_Black_24_BRH600226_D	No treatment	50	Female	N/A	Black
SA005052	MaleVsFemale_NT_Female_Caucasian_24_BRH600211_A	No treatment	50	Female	N/A	Caucasian
SA005053	MaleVsFemale_NT_Female_Caucasian_24_BRH600211_B	No treatment	50	Female	N/A	Caucasian
SA005054	MaleVsFemale_NT_Female_Caucasian_23_BRH600228_B	No treatment	50	Female	N/A	Caucasian
SA005055	MaleVsFemale_NT_Female_Caucasian_23_BRH600228_A	No treatment	50	Female	N/A	Caucasian
SA005056	MaleVsFemale_NT_Female_Caucasian_23_BRH600225_C	No treatment	50	Female	N/A	Caucasian
SA005057	MaleVsFemale_NT_Female_Caucasian_23_BRH600225_D	No treatment	50	Female	N/A	Caucasian
SA005058	MaleVsFemale_NT_Female_Caucasian_24_BRH600215_C	No treatment	50	Female	N/A	Caucasian
SA005059	MaleVsFemale_NT_Female_Caucasian_24_BRH600215_D	No treatment	50	Female	N/A	Caucasian
SA005060	MaleVsFemale_NT_Female_Caucasian_24_BRH600223_B	No treatment	50	Female	N/A	Caucasian
SA005061	MaleVsFemale_NT_Female_Caucasian_24_BRH600217_D	No treatment	50	Female	N/A	Caucasian
SA005062	MaleVsFemale_NT_Female_Caucasian_24_BRH600229_A	No treatment	50	Female	N/A	Caucasian
SA005063	MaleVsFemale_NT_Female_Caucasian_24_BRH600229_B	No treatment	50	Female	N/A	Caucasian
SA005064	MaleVsFemale_NT_Female_Caucasian_24_BRH600217_C	No treatment	50	Female	N/A	Caucasian
SA005065	MaleVsFemale_NT_Female_Caucasian_23_BRH600224_D	No treatment	50	Female	N/A	Caucasian
SA005066	MaleVsFemale_NT_Female_Caucasian_24_BRH600223_A	No treatment	50	Female	N/A	Caucasian
SA005067	MaleVsFemale_NT_Female_Caucasian_23_BRH600218_A	No treatment	50	Female	N/A	Caucasian
SA005068	MaleVsFemale_NT_Female_Caucasian_23_BRH600224_C	No treatment	50	Female	N/A	Caucasian
SA005069	MaleVsFemale_NT_Female_Caucasian_22_BRH600221_D	No treatment	50	Female	N/A	Caucasian
SA005070	MaleVsFemale_NT_Female_Caucasian_22_BRH600221_C	No treatment	50	Female	N/A	Caucasian
SA005071	MaleVsFemale_NT_Female_Caucasian_23_BRH600213_D	No treatment	50	Female	N/A	Caucasian
SA005072	MaleVsFemale_NT_Female_Caucasian_23_BRH600213_C	No treatment	50	Female	N/A	Caucasian
SA005073	MaleVsFemale_NT_Female_Caucasian_23_BRH600218_B	No treatment	50	Female	N/A	Caucasian
SA005074	MaleVsFemale_NT_Female_Caucasian_23_BRH600214_B	No treatment	50	Female	N/A	Caucasian
SA005075	MaleVsFemale_NT_Female_Caucasian_23_BRH600214_A	No treatment	50	Female	N/A	Caucasian
SA005076	MaleVsFemale_NT_Female_Hispanic_23_BRH600210_B	No treatment	50	Female	N/A	Hispanic
SA005077	MaleVsFemale_NT_Female_Hispanic_23_BRH600212_D	No treatment	50	Female	N/A	Hispanic
SA005078	MaleVsFemale_NT_Female_Hispanic_24_BRH600220_A	No treatment	50	Female	N/A	Hispanic
SA005079	MaleVsFemale_NT_Female_Hispanic_24_BRH600222_D	No treatment	50	Female	N/A	Hispanic
SA005080	MaleVsFemale_NT_Female_Hispanic_23_BRH600212_C	No treatment	50	Female	N/A	Hispanic
SA005081	MaleVsFemale_NT_Female_Hispanic_24_BRH600220_B	No treatment	50	Female	N/A	Hispanic
SA005082	MaleVsFemale_NT_Female_Hispanic_24_BRH600222_C	No treatment	50	Female	N/A	Hispanic
SA005083	MaleVsFemale_NT_Female_Hispanic_23_BRH600210_A	No treatment	50	Female	N/A	Hispanic
SA005084	MaleVsFemale_NT_Male_Caucasian_21_BRH600204_D	No treatment	50	Male	N/A	Caucasian
SA005085	MaleVsFemale_NT_Male_Caucasian_22_BRH600190_A	No treatment	50	Male	N/A	Caucasian
SA005086	MaleVsFemale_NT_Male_Caucasian_21_BRH600204_C	No treatment	50	Male	N/A	Caucasian
SA005087	MaleVsFemale_NT_Male_Caucasian_21_BRH600202_D	No treatment	50	Male	N/A	Caucasian
SA005088	MaleVsFemale_NT_Male_Caucasian_21_BRH600202_C	No treatment	50	Male	N/A	Caucasian
SA005089	MaleVsFemale_NT_Male_Caucasian_22_BRH600190_B	No treatment	50	Male	N/A	Caucasian
SA005090	MaleVsFemale_NT_Male_Caucasian_22_BRH600194_D	No treatment	50	Male	N/A	Caucasian
SA005091	MaleVsFemale_NT_Male_Caucasian_22_BRH600199_C	No treatment	50	Male	N/A	Caucasian
SA005092	MaleVsFemale_NT_Male_Caucasian_22_BRH600199_D	No treatment	50	Male	N/A	Caucasian
SA005093	MaleVsFemale_NT_Male_Caucasian_22_BRH600198_B	No treatment	50	Male	N/A	Caucasian
SA005094	MaleVsFemale_NT_Male_Caucasian_22_BRH600198_A	No treatment	50	Male	N/A	Caucasian
SA005095	MaleVsFemale_NT_Male_Caucasian_21_BRH600201_A	No treatment	50	Male	N/A	Caucasian
SA005096	MaleVsFemale_NT_Male_Caucasian_22_BRH600194_C	No treatment	50	Male	N/A	Caucasian
SA005097	MaleVsFemale_NT_Male_Caucasian_21_BRH600201_B	No treatment	50	Male	N/A	Caucasian
SA005098	MaleVsFemale_NT_Male_Caucasian_20_BRH600193_B	No treatment	50	Male	N/A	Caucasian
SA005099	MaleVsFemale_NT_Male_Caucasian_20_BRH600193_A	No treatment	50	Male	N/A	Caucasian
SA005100	MaleVsFemale_NT_Male_Caucasian_21_BRH600197_D	No treatment	50	Male	N/A	Caucasian
SA005101	MaleVsFemale_NT_Male_Caucasian_20_BRH600196_B	No treatment	50	Male	N/A	Caucasian
SA005102	MaleVsFemale_NT_Male_Caucasian_20_BRH600196_A	No treatment	50	Male	N/A	Caucasian
SA005103	MaleVsFemale_NT_Male_Caucasian_21_BRH600192_A	No treatment	50	Male	N/A	Caucasian
SA005104	MaleVsFemale_NT_Male_Caucasian_21_BRH600192_B	No treatment	50	Male	N/A	Caucasian
SA005105	MaleVsFemale_NT_Male_Caucasian_21_BRH600197_C	No treatment	50	Male	N/A	Caucasian
SA005106	MaleVsFemale_NT_Male_Hispanic_21_BRH600209_C	No treatment	50	Male	N/A	Hispanic
SA005107	MaleVsFemale_NT_Male_Hispanic_21_BRH600205_B	No treatment	50	Male	N/A	Hispanic
SA005108	MaleVsFemale_NT_Male_Hispanic_22_BRH600203_B	No treatment	50	Male	N/A	Hispanic
SA005109	MaleVsFemale_NT_Male_Hispanic_21_BRH600191_B	No treatment	50	Male	N/A	Hispanic
SA005110	MaleVsFemale_NT_Male_Hispanic_21_BRH600209_D	No treatment	50	Male	N/A	Hispanic
SA005111	MaleVsFemale_NT_Male_Hispanic_22_BRH600195_C	No treatment	50	Male	N/A	Hispanic
SA005112	MaleVsFemale_NT_Male_Hispanic_21_BRH600191_A	No treatment	50	Male	N/A	Hispanic
SA005113	MaleVsFemale_NT_Male_Hispanic_22_BRH600195_D	No treatment	50	Male	N/A	Hispanic
SA005114	MaleVsFemale_NT_Male_Hispanic_22_BRH600200_A	No treatment	50	Male	N/A	Hispanic
SA005115	MaleVsFemale_NT_Male_Hispanic_22_BRH600200_B	No treatment	50	Male	N/A	Hispanic
SA005116	MaleVsFemale_NT_Male_Hispanic_22_BRH600203_A	No treatment	50	Male	N/A	Hispanic
SA005117	MaleVsFemale_NT_Male_Hispanic_21_BRH600205_A	No treatment	50	Male	N/A	Hispanic
SA005118	MaleVsFemale_NT_Male_Hispanic_20_BRH600206_C	No treatment	50	Male	N/A	Hispanic
SA005119	MaleVsFemale_NT_Male_Hispanic_20_BRH600208_D	No 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 GCMS, 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