OMNImet •GUT: An at-home collection and ambient temperature transport device for fecal metabolomics®

Olle M. de Bruin1, Héloïse Breton1, Elizaveta Freinkman2, Anne M. Evans2, Suchitra K. Hourigan3,4, Evgueni Doukhanine1
1DNA Genotek Inc., Ottawa, ON, Canada, 2Metabolon, Inc., Durham, NC, USA, 3Inova Children’s Hospital, Falls Church, VA, USA, 4Pediatric Specialists of Virginia, Fairfax, VA, USA

Introduction

Short-chain fatty acids (SCFAs) are stable in OMNImet•GUT for at least 7 days at room temperatureResults (continued)

Metabolomic analysis of feces is a key methodology for understanding the functional rolesof microbial organisms and their interactions within the gut. To date, practical challenges
surrounding the collection and stabilization of fecal samples from human volunteers have
hampered large-scale metabolomic analyses. In response to the need for a collectionand stabilization solution for fecal metabolites, DNA Genotek® has developed theOMNImet®•GUT at-home collection kit. This kit includes a device featuring a volumetricallyvalidated sample input, a built-in mixing ball for rapid homogenization of the collected
sample and a metabolite-stabilizing solution.

SCFAs are key mediators of gut microbial activity and play important roles in regulating the immune system, maintaining the gut epithelial
Isobutyrate
Isobutyrate
Isobutyrate
2-Methylbutyrate
2-Methylbutyrate
2-Methylbutyrate
Acetate Acetate
Acetatebarrierand preventing
disorders such as metabolic
syndrome,
inflammatory
bowel disease and certain types of cancer.3• A high degree of fidelity (Spearman correlation 0.89) was observed between flash-frozen andOMNImet•GUT collected samples, which were immediately frozen (T0).
• Spearman correlations to flash-frozen raw feces remained in the 0.86-0.88 range after 1, 4 and
7 days of room temperature storage in OMNImet•GUT, indicating sustained metabolite stabilization
within the device.
• Amino acid and xenobiotic superpathways, which contain most of the known bioactive microbial
metabolites, displayed median Spearman coefficients in the 0.9 range at all time points (T0 to T7)
in OMNImet•GUT relative to matched flash-frozen samples.

Individual donor metabolomic profiles are maintained aftercollection in OMNImet•GUTMethodology
SCFA

7 donors (individual)

Pooled samples (high diversity)

Room temperature

OMNImet•GUT

Principal component analysis (PCA, top) and hierarchical clustering analysis (HCA, bottom) on
matched samples from 7 donors that had been flash-frozen or stored at room temperature in
OMNImet•GUT for varying lengths of time were performed to assess the impact of storing samples
in OMNImet•GUT on the ability to derive biological insights from human subject data.

Room temperature

Unstabilized

OMNImet•GUT

Unstabilized

Superpathway:

Experimental design for untargeted metabolomics
and for targeted SCFA analysis is depicted on the right.
For the freeze-thaw (FT) condition, an aliquot from each
OMNImet•GUT (ME-200) tube was held at -20°C for 7 days,
followed by 4 hours at 30°C. After the indicated storage
condition, all samples were held at -80°C until analysis. For
untargeted metabolomics, samples were subjected to drying
followed by automated biochemical extraction and analysis
by liquid chromatography and high resolution tandem
mass spectrometry (LC-MS/MS) on Metabolon’s Global
Platform.1,2 For SCFA analysis, feces pooled from 7 donors
was stored at room temperature for varying lengths of timein OMNImet•GUT and in an unstabilized condition, followedby targeted, quantitative analysis of eight SCFAs. Each tube
was centrifuged and an aliquot of the supernatant wasderivatized with 2,4-difluorophenyl hydrazine in presence ofinternal standards. Diluted derivatization reaction mixtures
were injected onto an Agilent 1290/AB Sciex QTRAP® 5500
LC-MS/MS system equipped with a C18 reversed phase
UHPLC column. The mass spectrometer was operated innegative mode using electrospray ionization (ESI).

Untargeted

Extraction
at T0

Relative Abundance:-4.00

Storage Condition:

Unstabilized

Unstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT

Unstabilized
OMNImet•GUT

Unstabilized Unstabilized

Unstabilized

Unstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT

Unstabilized
OMNImet•GUT

2-Methylbutyrate
2-Methylbutyrate
2-Methylbutyrate
OMNImet•GUT
OMNImet•GUT OMNImet•GUT

Acetate Acetate
AcetateOMNImet•GUT
OMNImet•GUT

Unstabilized Unstabilized

Unstabilized
OMNImet•GUUnstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT

Unstabilized
OMNImet•GU

time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)time (d)Propionate
PropionatePropionate

ButyrateButyrate

Unstabilized Unstabilized
Unstabilized
OMNImet•GUT
OMNImet•GUT OMNImet•GUT
Propionate
Propionate
Propionate

Unstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT
Butyrate
ButyrateButyrate

Unstabilized
OMNImet•GUT

Unstabilized Unstabilized
Unstabilized
OMNImet•GUT
OMNImet•GUT OMNImet•GUHexanoate
Hexanoate
Hexanoate

Unstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT

Unstabilized
OMNImet•GUT

Unstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT

Unstabilized Unstabilized
OMNImet•GUT
OMNImet•GUT

time (d)4.00

Unstabilized Unstabilized

Isobutyrate
Isobutyrate
Isobutyrate
OMNImet•GUT
OMNImet•GUT OMNImet•GUT

time (d)Unstabilized
OMNImet•GUT

time (d)time (d)time (d)Butyrate

HexanoateHexanoate Hexanoate

time (d)time (d)time (d)time (d)PARAM_GROUP_DESCRIPTION

timeWhile(d)time (d)(d)
time (d) twofoldtime (d)time (d)timeof(d)SCFAs in raw samples increased time
timetime
concentrations
approximately
to(d)tenfold after 4 days at room temperature,
concentrationsof(d)Fresh frozen

Freeze-thaw

T0
T1 T4
T4
T7 T7

P186 F/T

OMNImet•GUTME-200

Freeze-Thaw

P186 T0

T0

Extraction
at T1

P186 T1
P186 T4(1Comp.2

P186 T7

SUPER_PATHWAY
Superpathway:

Amino AcidDonors(1-7):

Stabilization of metabolites of infant stool collected in OMNImet•GUT

1
Lipid 2

56

Cofactors and Vitamins
EnergyNucleotide

3Peptide4
Xenobiotics

7

Partially Characterized Molecules

Donor ID:%)

Superpathway:

Results

Donor 7

Relative Abundance:
-4.00

4.00

Storage Condition:

PARAM_GROUP_DESCRIPTION
Fresh frozen

Flash-frozen
Relative Abundance:
P186 F/T
Freeze-thaw
Freeze-Thaw
P186 T0
-4.00
4.00
T0T0
PARAM_GROUP_DESCRIPTION
Storage Condition:
P186 T1
T1
T1
Fresh frozen
P186 T4T4
P186 F/TT4
Freeze-Thaw
P186
P186 T0 T7 T7
T0 T7
P186 T1

T1

Accurate measurement of individual metabolites and metabolic pathways aftercollection of fecal samples in OMNImet•GUT

P186 T4
SUPER_PATHWAY
Superpathway:
T4OMNImet•GUTME-200

Extraction
at T13.33

Com

Storage Condition:
Donor ID:

(20Superpathway:

p.1

p.
3

(1Com

Donor 1
Donor 2
Donor 3
Donor 4Donor 5
Donor 6ME-200

3.

97%)

Extraction
at T7

P186 T7Amino Acid

A hospital-based field test was conducted
where matched samples (n = 16 infant donors,each donor sampled at 2 time points; meantime between samples = 23 days, range 5-44days) were both flash-frozen and stored in
OMNImet•GUT tubes for 3-4 days separately.4
In the PCA of the 1,064 metabolites detectedin both flash-frozen and OMNImet•GUT tubes,
samples from the same individuals clusteredclosely regardless of collection method. For bothcollection methods there was a partial separation
between samples from the 2 time points, whichis consistent with both methods being able touncover time-dependent metabolomic changes
that occurred across patients during their gut
development.

T7Groups by color

30

Flash-frozen

20OMNImet•GUT10Comp.2 (13.70%)

Carbohydrate8.60%)

Extraction
at T4

SCFAs in OMNImet•GUT samples remained unchanged between 0 and 13 days at room temperature. Concentrations of isovalerate and valerateover the same period of time displayed a similar stability pattern (data not shown). These results indicate that OMNImet•GUT quenches bacterial
metabolism and that SCFAs are stable in the device for at least 13 days at room temperature.

Groups by shape0Time point 1-10

Time point 2-20-30-30		-20-10010203040

The samples arelabelledwith subject IDs 1-16

Comp.1 (18.79%)
Principal component analysis (PCA) of Frozen vs. OMNImet•GUT fecal samples collected at 2 time points.
The PCA was generated using 1,064 metabolites detected in both the Frozen and OMNImet•GUT tubes.

Carbohydrate

SUPER_PATHWAY
Superpathway:

Amino Acid
Cofactors and Vitamins
Carbohydrate

EnergyCofactors and Vitamins

Multi-donor stability: Correlation analysis (N = 7)

Lipid

Nucleotide

Nucleotide

Partially Characterized MoleculesPartially Characterized MoleculesPeptide

Xenobiotics

Xenobiotics

Donor
ID: ID:
DonorDonor 1
Donor 1
Donor 2
2
DonorDonor
3
DonorDonor
4
3
Donor 5
Donor 4
Donor 6

Storage
StorageCondition:
Condition:
Donor
Donor ID:
ID:

Storage Condition:Donor ID:

Donor 5
Donor 6

Donor 7

Donor 7

The results presented here demonstrate the benefits of OMNImet•GUT:• Convenient at-home collection of fecal samples for targeted and untargeted metabolomics• Stabilization of the gut metabolomic profiles for 7 days at room temperature• No time-dependent trends were consistently observed in either the PCA or the HCA, indicatingthat storage in OMNImet•GUT for up to 7 days at room temperature preserves the uniquemetabolomic fingerprint of human fecal samples.

• LC-MS/MS compatibility (including Metabolon’s Global Platform and SCFAs)

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Superior samples • Proven performanceConclusions• In both the PCA and the HCA, samples clustered clearly by donor, while differences amongstorage conditions and time points were minor relative to inter-donor differences.
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Ambient temperature collection and stabilization of infant stool in the OMNImet•GUT device yielded comparable results to flash-freezingin terms of the distinct metabolomic profiles of subjects and the biochemical signature of microbiome development over time.4

PeptideAssessment of the fidelity and stabilityof metabolomic profiles fromOMNImet•GUT samples was performedby calculating Spearman correlations
for all detected metabolites between
flash-frozen (FF) samples and matched
OMNImet•GUT samples held for 0, 1, 4and 7 days at room temperature from7 donors.

Energy Lipid• No cold chain required for shipping, handling or storage• Suitable for the collection and analysis of fecal samples from donors of any age, including infants

1 Evans, A.M., Bridgewater, B.R., Liu, Q., Mitchell, M.W., Robinson, R.J., et al. (2014). High resolution mass spectrometry improves data quantity and quality as compared to unit mass resolution mass spectrometry in high-throughput profiling metabolomics. Metabolomics 4: 132. http://doi.org/10.4172/2153-0769.1000132
2 Ford, L., Kennedy, A.D., Goodman, K.D., Pappan, K.L., Evans, A.M., Miller, L.A.D., Wulff, J.E., Wiggs, B.R., Lennon, J.J., Elsea, S., et al. (2020). Precision of a clinical metabolomics profiling platform for use in the identification of inborn errors of metabolism. J Appl Lab Med 5, 342-356. http://doi.org/10.1093/jalm/jfz026
3 Tan, J., McKenzie, C., Potamitis, M., Thorburn, A.N., Mackay, C.R., Macia, L. The role of short-chain fatty acids in health and disease. Adv Immunol. 2014;121:91-119. https://doi.org/10.1016/B978-0-12-800100-4.00003-9
4 Ramamoorthy, S., Levy, S., Mohamed, M., et al. An ambient-temperature storage and stabilization device performs comparably to flash-frozen collection for stool metabolomics in infants. BMC Microbiol 21, 59 (2021). https://doi.org/10.1186/s12866-021-02104-6
OMNImet®•GUT is for research use only, not for use in diagnostic procedures. Patent (www.dnagenotek.com/legalnotices) OMNImet and DNA Genotek are registered trademarks of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners. MK-01844 Issue 1/2021-07

www.dnagenotek.com • support@dnagenotek.com

Unstabilized
OMNImet•GU