Reference standardization was developed to address quantification and harmonization challenges for high-resolution metabolomics (HRM) data collected across different studies or analytical methods. Reference standardization relies on the concurrent analysis of calibrated pooled reference samples at predefined intervals and enables a singlestep batch correction and quantification for high-throughput metabolomics. Here, we provide quantitative measures of approximately 200 metabolites for each of three pooled reference materials (220 metabolites for Qstd3, 211 metabolites for CHEAR, 204 metabolites for NIST1950) and show application of this approach for quantification supports harmonization of metabolomics data collected from 3677 human samples in 17 separate studies analyzed by two complementary HRM methods over a 17-month period. The results establish reference standardization as a method suitable for harmonizing largescale metabolomics data and extending capabilities to quantify large numbers of known and unidentified metabolites detected by high-resolution mass spectrometry methods.

Untreated maple syrup urine disease (MSUD) results in mental and physical disabilities and often leads to neonatal death. Newborn-screening programs, coupled with the use of protein-modified diets, have minimized the severity of this phenotype and allowed affected individuals to develop into productive adults. Although inheritance of MSUD adheres to rules for single-gene traits, mutations in the genes for E1alpha, E1beta, or E2 of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex can cause the disease. Randomly selected cell lines from 63 individuals with clinically diagnosed MSUD were tested by retroviral complementation of branched-chain alpha-ketoacid dehydrogenase activity to identify the gene locus for mutant alleles. The frequencies of the mutations were 33% for the E1alpha gene, 38% for the E1beta gene, and 19% for the E2 gene. Ten percent of the tested cell lines gave ambiguous results by showing no complementation or restoration of activity with two gene products. These results provide a means to establish a genotype/phenotype relationship in MSUD, with the ultimate goal of unraveling the complexity of this single-gene trait. This represents the largest study to date providing information on the genotype for MSUD.

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Nellis, Mary M., Christopher B. Doering, Andrea Kasinski, and Dean J. Danner. Insulin increases branchedchain ␣-ketoacid dehydrogenase kinase expression in Clone 9 rat cells. Am J Physiol Endocrinol Metab 283: E853-E860, 2002. First published June 3, 2002 10.1152/ajpendo.00133.2002The branched-chain amino acids (BCAA) are committed to catabolism by the activity of the branched-chain ␣-ketoacid dehydrogenase (BCKD) complex. BCKD activity is regulated through the action of the complex-specific BCKD kinase that phosphorylates two serine residues in the E1␣ subunit. Greater BCKD kinase expression levels result in a lower activity state of BCKD and thus a decreased rate of BCAA catabolism. Activity state varies among tissues and can be altered by diet, exercise, hormones, and disease state. Within individual tissues, the concentration of BCKD kinase reflects the activity state of the BCKD complex. Here we investigated the effects of insulin, an important regulator of hepatic metabolic enzymes, on BCKD kinase expression in Clone 9 rat cells. Insulin effected a twofold increase in message levels and a twofold increase in BCKD kinase protein levels. The response was completely blocked by treatment with LY-294002 and partially blocked by rapamycin, thus demonstrating a dependence on phosphatidylinositol 3-kinase and mTOR function, respectively. These studies suggest that insulin acts to regulate BCAA catabolism through stimulation of BCKD kinase expression.hormone-controlled gene expression; branched-chain amino acids THE BRANCHED-CHAIN amino acids (BCAA), leucine, isoleucine, and valine, are classified as essential components of the mammalian diet because they cannot be synthesized de novo. BCAA account for up to 20% of the residues in the average protein. Catabolism of the BCAA provides energy, and their products serve as precursors for fatty acid synthesis. BCAA can modify other metabolic processes through a poorly understood mechanism whereby cells sense the concentration of these amino acids. Protein turnover, especially in the liver and muscles, is slowed when the BCAA concentration is maintained (6,12,32,37,38).Another player in the cellular regulation of protein turnover is insulin, which stimulates protein synthesis, especially in liver (11,15). A combination of insulin and leucine may be needed for the promotion of protein synthesis, and this action may occur through separate pathways to enhance assembly of the translation initiation complex (2, 5, 22). As expected, because tissues respond differently to insulin exposure, tissue-specific variation in these effects is reported (9,19,35,40).Maintenance of cellular BCAA concentration in mammals results from a balance between supply and catabolic loss. Supply results from dietary intake or breakdown of endogenous protein. Irreversible loss results from catabolism of the BCAA that begins with their reversible transamination to yield the branchedchain ␣-ketoacids (BCKA). Oxidative decarboxylation of the BCKA commits these compounds to their catabolic fate. The react...

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