Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA330453

Local Sample ID:	DS1-28-35
Subject ID:	SU003159
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

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Subject:

Subject ID:	SU003159
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
DS1-28-35	SA330453	FL039095	On-diet	Factor

Collection:

Collection ID:	CO003152
Collection Summary:	Blood samples were collected from consented study subjects and plasma isolated according to standard clinical laboratory protocols.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR003168
Treatment Summary:	This study was a clinical trial (NCT02653092) and was approved by the Colorado Multiple Institutional Review Board (COMIRB). All participants provided informed consent. Women aged 18-38 with menstrual cycles 25-35 days in length and no use of reproductive hormones within 3 months of enrollment were eligible for the study. Additional eligibility criteria included: a) no history of chronic disease affecting hormone production, metabolism, or clearance and no use of medications known to interact with reproductive hormones or insulin metabolism; b) normal screening prolactin and TSH; c) normal hemoglobin A1c; d) hemoglobin > 11 g/dl at screening; e) use of reliable non-hormonal contraception throughout the study period. Because the goal was to investigate the effects of a high-fat diet, women with a baseline dietary assessment indicative of >40% calories from fat were excluded, as were women who were unable to comply with the protocol, which involved eating only foods provided by the Colorado Clinical and Translational Sciences Institute’s (CCTSI) Nutrition Services. Because of the restrictions on their ability to consume animal or dairy fats, vegans and lactose intolerant individuals were excluded. Cycle 1 was a baseline cycle; Cycle 2 entailed consumption of the high-fat diet which was continued until frequent blood sampling was completed in the early follicular phase of Cycle 3. Cycle 3 was the immediate post-high-fat-diet cycle and Cycle 4 was a recovery cycle. Participants underwent baseline and end-of-diet assessments of gonadotropin dynamics. A 6-hour frequent blood sampling study was performed to determine endogenous LH and FSH secretion (Time 0-240 minutes) and concluded with a physiologic bolus of 75 ng/kg GnRH to assess pituitary responsiveness (Time 240-360 minutes). Frequent sampling studies for gonadotropin determination were all performed in the early follicular phase of the menstrual cycle (cycle days 2-5). Participants were also seen weekly during the high-fat diet cycle and had a blood draw to assess compliance. High-fat diet protocol. Participants completed a food preference screening survey and a 3-day diet diary, which estimated their daily caloric intake and approximate proportion of daily calories derived from fat. Participants were provided with a customized eucaloric high-fat diet comprised of approximately 48% calories from fat, 33% calories from carbohydrates and 19% calories from protein. Food was portioned into individual meals with snacks and boxed into 3-4 days’ worth at a time. Participants picked up food directly from the Clinical and Translational Research Center; when this was not possible, study staff delivered food to the participant. Urinary ketones were measured at each frequent blood sampling session (Siemens Healthineers, Malvern, PA). During administration of the high-fat diet, blood samples were drawn weekly for metabolomic analysis to determine compliance with the protocol.

Treatment ID:

TR003168

Treatment Summary:

This study was a clinical trial (NCT02653092) and was approved by the Colorado Multiple Institutional Review Board (COMIRB). All participants provided informed consent. Women aged 18-38 with menstrual cycles 25-35 days in length and no use of reproductive hormones within 3 months of enrollment were eligible for the study. Additional eligibility criteria included: a) no history of chronic disease affecting hormone production, metabolism, or clearance and no use of medications known to interact with reproductive hormones or insulin metabolism; b) normal screening prolactin and TSH; c) normal hemoglobin A1c; d) hemoglobin > 11 g/dl at screening; e) use of reliable non-hormonal contraception throughout the study period. Because the goal was to investigate the effects of a high-fat diet, women with a baseline dietary assessment indicative of >40% calories from fat were excluded, as were women who were unable to comply with the protocol, which involved eating only foods provided by the Colorado Clinical and Translational Sciences Institute’s (CCTSI) Nutrition Services. Because of the restrictions on their ability to consume animal or dairy fats, vegans and lactose intolerant individuals were excluded. Cycle 1 was a baseline cycle; Cycle 2 entailed consumption of the high-fat diet which was continued until frequent blood sampling was completed in the early follicular phase of Cycle 3. Cycle 3 was the immediate post-high-fat-diet cycle and Cycle 4 was a recovery cycle. Participants underwent baseline and end-of-diet assessments of gonadotropin dynamics. A 6-hour frequent blood sampling study was performed to determine endogenous LH and FSH secretion (Time 0-240 minutes) and concluded with a physiologic bolus of 75 ng/kg GnRH to assess pituitary responsiveness (Time 240-360 minutes). Frequent sampling studies for gonadotropin determination were all performed in the early follicular phase of the menstrual cycle (cycle days 2-5). Participants were also seen weekly during the high-fat diet cycle and had a blood draw to assess compliance. High-fat diet protocol. Participants completed a food preference screening survey and a 3-day diet diary, which estimated their daily caloric intake and approximate proportion of daily calories derived from fat. Participants were provided with a customized eucaloric high-fat diet comprised of approximately 48% calories from fat, 33% calories from carbohydrates and 19% calories from protein. Food was portioned into individual meals with snacks and boxed into 3-4 days’ worth at a time. Participants picked up food directly from the Clinical and Translational Research Center; when this was not possible, study staff delivered food to the participant. Urinary ketones were measured at each frequent blood sampling session (Siemens Healthineers, Malvern, PA). During administration of the high-fat diet, blood samples were drawn weekly for metabolomic analysis to determine compliance with the protocol.

Sample Preparation:

Sampleprep ID:	SP003165
Sampleprep Summary:	Lipids from 10 uL of plasma were extracted with 90 uL of cold methanol. Samples were briefly vortexed at room temp then incubated at -20 C for 30 min. Supernatants were clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials.
Processing Storage Conditions:	4℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN004993	AN004994
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	NEGATIVE	POSITIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH003772
Chromatography Summary:	Negative C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-3 min 10-95% B at 0.3 mL/min, 3-4.2 min hold at 95% B at 0.3 mL/min, 4.2-4.3 min drop to 10% B at 0.45 mL/min, 4.3-4.9 min hold at 10%B while lowering flow rate to 0.4 mL/min, 4.9-5 min hold at 10%B while lowering flow rate to 0.3 mL/min.
Flow Rate:	0.3-0.4 ml/min
Sample Injection:	10 uL
Solvent A:	75% water/25% acetonitrile; 5 mM ammonium acetate
Solvent B:	90% isopropanol/10% acetonitrile; 5 mM ammonium acetate
Chromatography Type:	Reversed phase

Chromatography ID:	CH003773
Chromatography Summary:	Positive C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-3 min 30-100% B at 0.3 mL/min, 3-4.2 min hold at 100% B at 0.3 mL/min, 4.2-4.3 min 100-30% B at 0.4 mL/min, 4.3-4.9 min hold at 30%B and 0.4 mL/min, 4.9-5 min hold at 30%B while lowering flow rate to 0.3 mL/min
Flow Rate:	0.3-0.4 ml/min
Sample Injection:	10 uL
Solvent A:	75% water/25% acetonitrile; 5 mM ammonium acetate
Solvent B:	90% isopropanol/10% acetonitrile; 5 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004733
Analysis ID:	AN004993
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control.
Ion Mode:	NEGATIVE

MS ID:	MS004734
Analysis ID:	AN004994
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control.
Ion Mode:	POSITIVE