Metabolic Research at Texas A&M University.

Metabolic research is performed at the 23,000 sq ft Center for Translational Research in Aging and Longevity (CTRAL, www.ctral.org) in the Department of Health and Kinesiology (HLKN) at Texas A&M in College Station, TX and is a state-of-the-art translational biomedical research facility to conduct exercise, nutrition and metabolism related studies. The CTRAL Clinical Research Unit (CRU) accommodates basic and applied clinical research in humans suitable for single-day, as well as multi-day 24/7 studies. The CTRAL Human Research Support Core houses a metabolic kitchen, a clean room for clinical preparations, a laboratory to facilitate STAT blood/tissue processing, a secured data archive system, a secured/controlled sample storage/biobank and a clinical waste management system. The CTRAL Metabolomics and Fluxomics Core is equipped with Mass Spectrometry and other analytical facilities, and the CTRAL Function Core houses a stress testing area, a body composition/ bone density assessment area, an isotonic and isokinetic strength testing area, and an anaerobic capacity analysis area. Projects at CTRAL are mainly focused on clinical nutrition, protein and amino acid metabolism and exercise. The main focus of research protocols include changes in the metabolism of amino acids, notably arginine, citrulline, glutamine and the branched-chain amino and keto acids as they contribute in lung disease, cancer, heart failure, gut function, cognitive decline, and critical illness. Also, projects are performed where muscle loss by 10 days bed rest in older adults is attenuated with special protein supplements. Glucose-clamping studies are also done to better understand the role of insulin resistance on muscle protein anabolism. Besides this, extensive experience and methodology are available to quantify the rate of endogenous de novo synthesis of arginine and to measure whole body arginine, citrulline, nitric oxide, protein kinetics and the kinetics of many other amino acids like methyl-histidine, hydroxyproline, and methionine in clinical conditions. We recently introduced the use of the pulse injection of stable isotopes to measure metabolism. This approach requires only 1 combined injection and sampling catheter, does not require priming, and will generate data that will identify different pools in the body. In addition, we follow metabolism after ingestion of deuterium-labeled water (D2O). D2O labels proteins, fatty acids, and nucleotides in a specific manner and this is used to calculate its synthesis for days to weeks. Our innovative techniques with stable isotopes and with advanced mass spectrometry systems play central roles in all our studies.

 

CTRAL Clinical Research Unit (CRU)

The CTRAL-CRU consists of 12 overnight stay beds (4 private rooms, 4 semi-private rooms, one 4-bedroom), 4 private exam/procedure rooms, 1 room to measure resting energy expenditure, a nurse station, family waiting room, phlebotomy room, offices, and several storage rooms. Two private rooms have bariatric size beds. Each bed has oxygen supply, nurse alarm system, emergency power, internet connection, direct point-to point communication possibility with nursing station, TVS/DVD, recliner for (overnight) visitors, personal storage for subjects. CTRAL-CRU has 8 Baxter 6201 infusion pumps, 5 custom made hot-boxes to obtain arterialized-venous blood samplings, automatic and manual B/P Machines, pulse oxygen meter, CPR board, blood glucose glucometers (Accu-check Aviva), and 3 wheelchairs. Research nursing staff and study physician are responsible for the medical aspects of the outpatient part of the studies. CTRAL is at the Texas A&M campus and is readily accessible with easy parking for study participants and staff. Wall mounted storage boxes for sample pick-up by commercial laboratories are also available.

 

CTRAL Recruitment and Compliance Core

The recruitment core of CTRAL is responsible for the recruitment of hospitalized, outpatients, and healthy volunteers. CTRAL PIs currently have successful research collaborations with many physicians in the Bryan/College Station area at College Station Medical Center, Baylor Scott and White Medical Center, CHI St. Joseph Health, and MD Anderson in Houston. The pulmonologist Drs. Harrykissoon and Zachria will be responsible for the recruitment of COPD subjects at College Station Medical Center; the pulmonologist Dr. Veley will do the same at Baylor Scott and White Medical Center in College Station. CTRAL PIs currently have good research collaboration with many physicians at the Scott and White clinic and have full support of the medical director from Baylor Scott and White Medical Center.  Our RNs have extensive experience at the medical centers in College Station. Currently, CTRAL has 8 active IRB approval studies (NCT02157844, NCT01173354, NCT01624792, NCT02770092, NCT01734473, NCT01154400, NCT02780219, NCT02065141) that continuously recruit stable COPD patients. The Compliance Core with very experienced staff is responsible that CTRAL activities are in compliance with institutional IRB, IACUC, Biosafety, environmental, health safety, and other federal regulations.

 

CTRAL Human Research Support Core

A metabolic kitchen (commercial grade) is available 24/7 for food/test diet/food supplement and non-hazardous oral compounding (USP 795) preparations for human subjects, and has a controlled/monitored 4°C and -20°C storage. A clean room unit for clinical preparations is present, containing an IV compounding facility (USP 797 grade) for e.g. non-hazardous stable isotope infusate compounding, a controlled/monitored 4°C and -20°C storage, and a sterilizer for clinical tools/instruments. This facility can also be used to prepare aseptic syringes and infusion bags on the clinical test days. In addition, an IV compounding outsource option is available with a licensed sterile compounding pharmacist Steve D. Mitchell, Pharm.D, Specialty Pharmacy Services, Inc., Temple, TX. Sterility and pyrogenicity of the prepared IV compounded infusates are tested by CTRAL according to guidelines of USP 797 and the standard operation protocol of the pharmacist. Experienced staff is available to ensure appropriate use of stable isotope technology in human subjects. A laboratory is present to facilitate STAT blood/ tissue processing and is equipped with 2 biosafety cabinets, 2 centrifuges and a blood cell counter. A high-density secured data archive system is located in the secured office area. A secured/ controlled/ monitored sample storage/ biobank is available with a CO2-backup system and emergency power for seven -80°C freezers with a capacity to store 250.000 samples. Clinical waste management exists consisting of a decontamination room for reusable tools/instruments used in human subjects, a soil room and a waste autoclave to sterilize clinical waste.

 

CTRAL Function Core

Major function equipment is available in the CTRAL Function Core and includes a NO breath analyser (NIOX), 4 FEV1 analyser (SpiroD), Handgrip dynamometer (Vernier), MicroRPM – MIP/MEP to measure mouth pressures (Carefusion), Resting energy expenditure using ventilated hood (ParvoMedics True One 2400), EKG (Nasitt Cardio System), Heart Rate Monitors for exercise (Polar E600), Pedometers (Sportline Model 370), Exercise Bike (Schwinn), Leg Press (Nebula), Exercise Bike – Watt Measurement (Sport excalibar), Bench Press (Nebula), Smith Press (Life Fitness), Stress Test Treadmills – Graded exercise testing (VO2 max) and KinCom Machine (KinCom) and resistance endurance and resistance rehabilitation Training Facilities with whole body workout circuit machines (leg extension/curl, squat, latissimus pulldown, chest press, biceps curl, triceps extension). Eight different thera-bands are available with increasing resistance which allows suitable resistance for each individual and a graded increase in workload during exercises. A change from one color to another results in an increased resistance of 20 to 30%. Body composition is measured by multiple techniques. Using the deuterium/bromide dilution technique, each study subject will consume a preweighed oral dose of deuterium labeled water (D2O) and sodium bromide after which both total body and extracellular water will be estimated from deuterium dilution and bromide space by measuring the D2O {Mahsut, 2011, 351-353}{Walker, 2015, 838-843} and Br levels in plasma or urine at CTRAL Analytical Labs. In addition, body composition at the whole-body level and within body compartments (extremities, trunk) are measured by means of a DXA system (Hologic QDR 4500W with Apex Software), thereby measuring fat tissue, lean tissue, and bone mineral content, and by Bioelectrical Impedance Analysis (BIA) D550 (Impedimed). Intra- and extramuscular fat and muscle content of the leg are measured with a cross-sectional CT scan of the mid-thigh, calculated with commercially available imaging software at the {Goodpaster, 2008, 1498-503}{Koster, 2011, 888-95}(NCT02090387) at the Texas A&M Institute for Preclinical Studies (0.6 mi from CTRAL) or at Bryan Radiology Associates, located 4.9 miles from CTRAL.

 

CTRAL Metabolomics and Fluxomics Core

This core features major instrumentation for LC/MS/MS, GC/MS/MS and clinical biochemistry; and additional major and minor equipment to prepare samples for such assays. For LC/MS/MS, Sciex (Foster City CA) 5500 QTRAP® and 3200 QTRAP® instruments are hybrid tandem mass spectrometers operable in all triple-quadrupole (QqQ) and quadrupole–linear ion trap (QqLIT) scan modes. Their TurboV® heated ESI and APCI sources deliver very high and moderately high ion fluxes, respectively; minimize interchannel delays by LINAC® patented axial ejection of fragment ions from the collision cells; acquire data in true ion-counting mode; and reduce noise by a patented algorithm that does not obscure trace signals. Therefore, it is feasible for CTRAL to simultaneously acquire concentration and enrichment information on scores of metabolic targets fully controlled by internal and external isotope-dilution standards. For less demanding LC/MS/MS assays, this core also houses an API 2000 triple quad with TurboIon source (Sciex). Any mass spec can be preceded by any of 4 UHPLC systems: for microanalytical (0.3-1.0 mm i.d. column) chromatography, Ekspert® microLC 200 and ExpressHT ultra® microanalytical-scale LC systems (Eksigent, part of Sciex) feature pulseless (non-reciprocating) binary pumps with high-pressure mixing that deliver eluent flow rates of 5-200 µL/min, and low-carryover PAL-HTC-xt-DLW and PAL-HTC autosamplers (CTC Analytics AG, Zwingen, Switzerland) both flexibly controllable by scripts written in-house; for assays resistant to scale down, two Prominence XR UFPLC systems (Shimadzu) permit classical analytical and semi-preparative scale HPLC. In addition to 3 Analyst® v. 1.6.3 acquisition software licenses for the 3 mass specs, CTRAL owns 3 licenses for MultiQuant® (Sciex) analysis software, unrivaled in its capacity to efficiently and accurately extract tabular data from mass chromatograms. For GC/MS/MS: there is a Scion TQ triple-quadrupole MS/MS interfaced with a 436 GC oven and a CP-8400 99-vial autosampler (Bruker Daltonics, Billerica MA). The system has a quick-cool (TPV) inlet, a backflush control valve, and both EI and +/- CI sources, the latter operable with either methane or ammonia collision gas (Bruker Daltonics Inc., Fremont CA). The existing 4 mass spectrometers occupy 4 of 6 clusters along two walls of a sound- and ventilation-isolated pump room accommodating roughing pumps, gasses and power conditioners; 2 fully equipped clusters stand vacant for future LC/MS/MS or GC/MS/MS systems.  Clinical biochemistry: the lab is equipped with a cobas c 111 analyzer (Roche Diagnostics, Indianapolis IN) and a Cell-Dyn 1800 analyzer (Abbott, Columbus, OH). Microplate spectrophotometry: e.g., to assay plasma proteins such as IL-6, TNF, and hsCRP, there is a plate reader:  ELx808 (BioTek, Winooski VT) and a Bio-Plex 200 (Bio-Rad, Hercules CA). Oxygraph equipment 2* Oroboros-2K: G0061 and F-0067, Sample processing equipment includes 2 Speed-Vac (Thermo) and 1 Zanntek Zip-Vac (Glas-Col) sample concentrators, 3 chilled  centrifuges (Megafuge 40R Hereaus, Eppendorf 5427R), a chilled high speed Lab Centrifuge (Hereaus Biofuge), semi-micro balance XP205 and MS303-S top-loading analytical balances (Mettler-Toledo, Columbus OH), shaking incubator S1500 (Stuart), 2 dry block heaters (VWR, Thermolyne), dry bath incubator (Fisher), titer plate shaker (Thermo), mini- bead beater (Biospec).  Support equipment includes a two-stage water treatment system using an AFS-8D Milli-Q analytical water purifier (Millipore) for general lab use and to feed a Barnstead Mega-Pure MP-1 Glass Still with Automatic Collection System (Thermo) providing water for exacting analyses, two instrument-gas (nitrogen, zero-air) generators (PEAK Scientific) supplied by a 30 hp air compressor system (Powerex) with dual coalescing dryers and an 80 G storage tank, a laboratory vacuum system (Edwards), an ice machine (Scotsman), oven (Binder), ultrasonic bath 3510 (Branson), pH meter (Fisher), stirrer (PC-420D: Corning) 4 vortexers (IKA), water bath (Fisher), incubator (Boekel), pipet aid (Drummond Scientific), bacti cinerator IV (McCormick Scientific), 3 biosafety cabinets (Esco/Nuance), Lindberg/Blue glassware furnace (Thermo), cell culture incubator (NuAire_NU-5810), inverted microscope (Nikon) for cell culture and two (1 large, 1 benchtop) autoclaves (Tuttnauer).

 

CTRAL computing facilities consists of 5 Brother printers, 5 Touch Smart HP computers, 35 DeskTop HP and Dell PC, 30 HP Chromebooks, 4 Projectors and state-of-the-art equipment for web meetings. In addition, software licenses for all PC with Adobe PDF and Microsoft Office. Separate licenses for SPSS statistical (3), STATA statistical, Graphpad Prism statistical and presentation Software (6), SAAM Software for compartmental analysis (1) and several specific equipment related software licenses. Also programming in R for specific data handling questions. CTRAL has 12 offices, 1 conference room, one break room, several storage rooms and the CTRAL-CRU, CTRAL Analysis and Function Cores with a total of 23,000 sqft.