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MB Sample ID: SA330478
Local Sample ID: | DS1-28-55 |
Subject ID: | SU003159 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Female |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
DS1-28-55 | SA330478 | FL039097 | Pre-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. |
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 |