Metabolic Syndrome in Bone Marrow Transplant Survivors

Specific aim 1: Surveillance for endocrinopathy

• We will test for defects in the growth hormone (GH) axis utilizing GH stimulation testing. Briefly the patient (subject) will have studies conducted in the fasted state using two independent secretagogues (arginine and clonidine) per procedures utilized in endocrine clinical practice (See appendix for protocol). GH deficiency will be identified in those subjects whose stimulated GH value is < 10 ng/dl at all time points, according to standard criteria. We will also evaluate IGF-1 and IGFBP-3 levels and results will be interpreted per age normal values. As part of our surveillance for GH axis defects, we will obtain a bone age X-ray. Results will be interpreted according to the standards of Greulich and Pyle.
• Sex steroid levels. Testing for estrogen and testosterone levels can be done on the same day and at the same time as the GH stimulation tests. Results will be compared to bone age matched normal values as published by Esoterix Laboratory (Calabasas Hills, CA).
• OGTT- A 2-hour oral glucose tolerance test (OGTT) will be conducted at or before 9 AM, following an overnight fast. To minimize discomfort, EMLA® cream will be placed on the site for intravenous catheter (IV) insertion for 30 minutes before the IV is placed. The IV will be placed in an antecubital vein by an experienced nurse. Baseline blood samples will be withdrawn from the IV. The patient will then drink a glucola (1.75 gms CHO/kg, max 75 gms) over 3 minutes. Blood samples will be withdrawn from the IV at 30, 60 and 120 minutes following Glucola ingestion. Blood will be analyzed immediately using an YSI glucose analyzer (Yellow Springs OH) for serum glucose levels. Glucose tolerance will be categorized according to World Health Organization17. Additional blood will be collected and frozen for later analysis of insulin levels to be analyzed in the PI's laboratory.
• A fasting blood sample will be obtained for later evaluation of a fasting lipid profile. This will be conducted in the NCH laboratory. Values will be analyzed according to published age normal values18.
• We will measure waist circumference and report according to pediatric normal values6.

Specific aim 2:

• Measurement of hepatic glucose production (HGP) and protein turnover. Patients will be asked to spend the night in the Clinical Study Center (arrive by 9 p.m.). He/she will remain NPO, except for sugar- and caffeine-free liquids after 9 p.m. Prior to going to sleep, using EMLA® cream, an IV catheter will be inserted into the antecubital vein. At 4:00 a.m. we will begin infusion of stable isotopes [1-13C] NaHCO3 (bolus 0.2 mg/kg then 0.6 mg/kg/hr), [1-13C] leucine (bolus 0.35 mg/kg followed by 0.65 mg/kg/hr) and 6,6-[2H2]glucose (bolus 2.5 grams/kg followed by constant infusion of 2.0 mg/kg/hour) for 3.0 hours (isotopic equilibration period). Blood will be drawn at baseline and again after isotopic equilibration. Samples will be centrifuged and the serum frozen for future analysis by mass spectrometry in the P.I.'s laboratory. We and others have previously described these methods.20 Additionally, we will assess resting energy expenditure (REE) by 30 minute hood indirect calorimetry using the VMax calorimeter (Viasys Healthcare, Yorba Linda, CA).
• Measurement of hepatic and peripheral insulin resistance using a hyperinsulinemic euglycemic clamp. We will measure hepatic and peripheral insulin sensitivity using a hyperinsulinemic euglycemic clamp in conjunction with continued 6,6-[2H2]glucose. Again using EMLA® cream, a second IV catheter will be inserted retrograde into the back of the opposite hand and the hand will be placed in a warming box. Steady state blood will be collected from this catheter.

At 7 a.m. we will begin the clamp. Insulin will be infused initially at 10 mU/m2/min. After at least 2 hours (or longer if steady state has not occurred), the insulin infusion rate will be increased to 40 mU/m2/min. The second dose will be continued for at least 2 hours, or longer until steady-state has occurred. During insulin infusion, the patient will also receive an infusion of 20% dextrose. The rate of dextrose infusion will be adjusted every 5 - 15 minutes to keep plasma glucose 88-95 mg/dl. Plasma glucose will be measured every 5-10 minutes at bedside using an automated glucose oxidase technique (Glucose Analyzer; YSI, Yellow Springs, OH). To prevent hypokalemia and hypophosphatemia, K2HPO4 will be infused throughout the study, and serum potassium levels will be measured at baseline and at the end of the study. Blood will be collected for later detection of 6H6 in plasma before infusion of isotope (baseline), at steady state (just before insulin infusion) and at the end of each insulin infusion "step." We will also collect blood for analysis of plasma insulin levels at these time points. Our group has previously reported the details of these methods21.