Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA056631

Local Sample ID:	ms6034-8
Subject ID:	SU000986
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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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
ms6034-8	SA056631	FL010256	135	Time point
ms6034-8	SA056631	FL010256	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.

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