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MB Sample ID: SA056734
Local Sample ID: | ms6243-6 |
Subject ID: | SU000986 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU000986 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
ms6243-6 | SA056734 | FL010264 | 60 | Time point |
ms6243-6 | SA056734 | FL010264 | control | Group |
Collection:
Collection ID: | CO000980 |
Collection Summary: | We will perform a hyperinsulinemic-euglycemic clamp as previously described except that in this current proposal we will perform the study for 3 hours.(36) The goal for this infusion is to assess the effect of hyperinsulinemia on lysine and AAA kinetics and whether metformin changes the effect of hyperinsulinemia. We will collect samples every 10 min for glucose. 40% dextrose will be infused at a variable rate during the clamp to maintain euglycemia.(36) During the last hour of the clamp, 5 blood samples will be drawn for measurement of stable isotope tracer enrichment and amino acid concentrations. |
Sample Type: | Blood (plasma) |
Treatment:
Treatment ID: | TR001000 |
Treatment Summary: | Ten women with PCOS will receive metformin therapy, and ten women will be randomized to receive no therapy and undergo the same repeat visits after 3 months. Ten age-matched women without PCOS with a BMI < 25 will be recruited as the control group for the baseline visit. Aside from criteria that they do not have PCOS and have a BMI < 25, this control group will have the same inclusion and exclusion criteria below. They will not receive metformin and will not return for repeat visits after 3 months. Metformin therapy: Previous studies with metformin demonstrated improvement in insulin sensitivity as early as 3 months with 1000 mg daily.(5, 33, 34) Metformin will be initiated with 500 mg extended-release tablet daily for one week, 1000 mg daily for one week and then 1500 mg daily. Visits 4 and 5 will be conducted three months after full dose is achieved. Oral glucose tolerance test: After 2 baseline fasting samples, 75 g of oral dextrose will be ingested with blood samples will be drawn at 10’, 20’, 30’, 60’, 90’, 120’, 150’and 180’ for measurement of glucose, insulin, c-peptide. Insulin sensitivity will be calculated using the oral glucose minimal model. Stable isotope tracer infusions (Figure 5): Three days prior to the tracer study, the participants will be placed on a weight-maintaining diet consisting of 50% carbohydrates, 20% protein, and 30% fats. Fat free mass (FFM) measured by dual-energy x-ray absorptiometry will be used for dose calculations of the stable isotope tracers and insulin infusions for the hyperinsulinemic-euglycemic clamp. A priming bolus dose of L-[α-15N]-lysine, 3 to 5 μmol/kg FFM, will be given at the start of a 3 hour infusion of 3 to 5 μmol/kg FFM/hr to achieve a plateau as previously described.(29) At the same time, a priming bolus of 1 to 2 μmol/kg FFM L-[1-13C]-2-aminoadipic acid will be given followed by infusion of 1 to 2 μmol/kg/hr based on prior study.(30) A retrograde hand intravenous line will be placed with the hand placed in a warm box maintained at 140°F to obtain arterialized venous blood samples for measurement of lysine and AAA concentrations and stable isotopic enrichment at steady state and during the clamp. Hyperinsulinemic-euglycemic clamp: We will perform a hyperinsulinemic-euglycemic clamp as previously described except that in this current proposal we will perform the study for 3 hours.(36) The goal for this infusion is to assess the effect of hyperinsulinemia on lysine and AAA kinetics and whether metformin changes the effect of hyperinsulinemia. We will collect samples every 10 min for glucose. 40% dextrose will be infused at a variable rate during the clamp to maintain euglycemia.(36) During the last hour of the clamp, 5 blood samples will be drawn for measurement of stable isotope tracer enrichment and amino acid concentrations. |
Sample Preparation:
Sampleprep ID: | SP000993 |
Sampleprep Summary: | Plasma lysine and AAA concentrations and isotopic enrichment will be analyzed as previous described via a Thermo Fisher Q Exactive plus mass spectrometer coupled with a Dionex UltiMate 3000 Binary RSLC liquid chromatograph. |
Combined analysis:
Analysis ID | AN001554 | AN001555 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
Column | Zorbax Extended C18 | Zorbax Extended C18 |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
Ion Mode | POSITIVE | POSITIVE |
Units | uM | MPE |
Chromatography:
Chromatography ID: | CH001090 |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Zorbax Extended C18 |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS001432 |
Analysis ID: | AN001554 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | uM |
Ion Mode: | POSITIVE |
MS ID: | MS001433 |
Analysis ID: | AN001555 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | MPE (molar percent excess ) |
Ion Mode: | POSITIVE |