To better understand the molecular mechanisms underpinning physiological variation in human populations, metabolic phenotyping approaches are increasingly being applied to studies involving hundreds and thousands of biofluid samples. Hyphenated ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) has become a fundamental tool for this purpose. However, the seemingly inevitable need to analyze large studies in multiple analytical batches for UPLC-MS analysis poses a challenge to data quality which has been recognized in the field. Herein, we describe in detail a fit-for-purpose UPLC-MS platform, method set, and sample analysis workflow, capable of sustained analysis on an industrial scale and allowing batch-free operation for large studies. Using complementary reversed-phase chromatography (RPC) and hydrophilic interaction liquid chromatography (HILIC) together with high resolution orthogonal acceleration time-of-flight mass spectrometry (oaTOF-MS), exceptional measurement precision is exemplified with independent epidemiological sample sets of approximately 650 and 1000 participant samples. Evaluation of molecular reference targets in repeated injections of pooled quality control (QC) samples distributed throughout each experiment demonstrates a mean retention time relative standard deviation (RSD) of <0.3% across all assays in both studies and a mean peak area RSD of <15% in the raw data. To more globally assess the quality of the profiling data, untargeted feature extraction was performed followed by data filtration according to feature intensity response to QC sample dilution. Analysis of the remaining features within the repeated QC sample measurements demonstrated median peak area RSD values of <20% for the RPC assays and <25% for the HILIC assays. These values represent the quality of the raw data, as no normalization or feature-specific intensity correction was applied. While the data in each experiment was acquired in a single continuous batch, instances of minor time-dependent intensity drift were observed, highlighting the utility of data correction techniques despite reducing the dependency on them for generating high quality data. These results demonstrate that the platform and methodology presented herein is fit-for-use in large scale metabolic phenotyping studies, challenging the assertion that such screening is inherently limited by batch effects. Details of the pipeline used to generate high quality raw data and mitigate the need for batch correction are provided.

Background-Combination therapy consisting of mechanical unloading using a left ventricular assist device (LVAD) and pharmacological intervention can promote recovery from end-stage heart failure, but the mechanism is unknown. Preliminary microarray analysis revealed a significant and unexpected decrease in myocardial arginine:glycine amidinotransferase (AGAT) gene expression during recovery in these patients. The aim of this study was to evaluate the expression and role of AGAT expression in heart failure and recovery. Methods and Results-We used quantitative real time (TaqMan) polymerase chain reaction to examine myocardial AGAT mRNA expression in implant and explant samples from recovering patients after combination therapy (nϭ12), end-stage heart failure (ESHF) samples from stable patients undergoing transplantation without LVAD support (nϭ10), and donor hearts with normal hemodynamic function (nϭ8). AGAT mRNA expression was significantly elevated in all heart failure patients relative to donors (4.3-fold [PϽ0.001] and 2.7-fold [PϽ0.005] in LVAD and ESHF relative to donors, respectively) and returned to normal levels after recovery. AGAT enzyme activity was detectable in both human and rat myocardia and was elevated in heart failure. Conclusions-Our data highlight local and potentially regulated expression of AGAT activity in the myocardium and suggest a specific response to heart failure involving elevated local creatine synthesis. These findings have implications both for the management of recovery patients undergoing combination therapy and for heart failure in general.

. Effects of chronic administration of clenbuterol on function and metabolism of adult rat cardiac muscle. Am J Physiol Heart Circ Physiol 288: H1468 -H1476, 2005. First published November 4, 2004 doi: 10.1152/ajpheart.00624.2004, a ␤2-agonist, is known to produce skeletal and myocardial hypertrophy. This compound has recently been used in combination with left ventricular assist devices for the treatment of end-stage heart failure to reverse or prevent the adverse effects of unloading-induced myocardial atrophy. However, the mechanisms of action of Clen on myocardial cells have not been fully elucidated. In an attempt to clarify this issue, we examined the effects of chronic administration of Clen on Ca 2ϩ handling and substrate preference in cardiac muscle. Rats were treated with either 2 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 Clen or saline (Sal) for 4 wk with the use of osmotic minipumps. Ventricular myocytes were enzymatically dissociated. Cells were field stimulated at 0.5, 1, and 2 Hz, and cytoplasmic Ca 2ϩ transients were monitored with the use of the fluorescent indicator indo-1 acetoxymethyl ester. Two-dimensional surface area and action potentials in current clamp were also measured. We found that in the Clen group there was significant hypertrophy at the organ and cellular levels compared with Sal. In Clen myocytes, the amplitude of the indo-1 ratio transients was significantly increased. Sarcoplasmic reticulum Ca 2ϩ content, estimated by rapid application of 20 mM caffeine, was significantly increased in the Clen group. The action potential was prolonged in the Clen group compared with Sal. Carbohydrate contribution to the tricarboxylic cycle (Krebs cycle) flux was increased several times in the Clen group. This increase was associated with decreased expression of peroxisome proliferator-activated receptor-␣. This study shows that chronic administration of Clen induces cellular hypertrophy and increases oxidative carbohydrate utilization together with an increase in sarcoplasmic reticulum Ca 2ϩ content, which results in increased amplitude of the Ca 2ϩ transients. These effects could be important when Clen is used in conjunction with left ventricular assist devices treatment.sarcoplasmic reticulum Ca 2ϩ content; tricarboxylic cycle; left ventricular assist devices LONG-TERM LEFT VENTRICULAR (LV) assist device (LVAD) support has been shown to improve survival and quality of life in patients with advanced heart failure and contraindications to cardiac transplantation (22). LVAD support is also routinely used as a bridge to transplantation in patients with end-stage heart failure. In a number of patients, with the use of this strategy in association with pharmacological therapy (combination therapy), a significant improvement in myocardial performance has been observed. In some cases the mechanical device could be explanted without resorting to heart transplantation ("bridge to recovery") (11-13, 27, 40).LVAD support results in profound and complex changes in the structure and function of the myocardium, which include "...