Extraction and analysis of carnitine and acylcarnitines by electrospray ionization tandem mass spectrometry directly from dried blood and plasma spots using a novel autosampler.

RATIONALE: Acylcarnitines are routinely analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) both in clinical diagnostic and public health newborn screening laboratories from plasma and dried whole blood spots (DBS) on filter paper. The use of DBS as a convenient method of collecting and storing samples for subsequent analysis of various biomolecules is increasing, thus prompting the development of new devices to recover and quantify such analytes in an automated manner. METHODS: Acylcarnitines were extracted directly from DBS using a novel autosampler that sequentially loads DBS cards into a pneumatic clamp and then pumps a fixed volume of solvent containing appropriate internal standards through a section of the DBS card directly into a triple quadrupole mass spectrometer via ESI. Plasma was first spiked with internal standard then spotted onto filter paper for analysis. RESULTS: Acylcarnitines were analyzed in DBS, and both free and total carnitine were assayed in dried plasma spots (DPS). Results using the new autosampling technique were of equal quality to those obtained by punching a 3-mm diameter disk from a DBS or DPS, then extracting and analyzing the target analytes from conventional 96-well microtiter plates, with far reduced time per sample. Recovery for most analytes was >60% and reproducibility was generally within 20% (CV). CONCLUSIONS: The simplicity and robustness of the DBS autosampler make it an attractive alternative to conventional methods of analyzing DBS specimens, thus saving time and labor costs, especially in high-throughput applications. Although the method as described is for direct infusion analysis, the autosampler is easily coupled to column hardware for applications requiring liquid chromatography/mass spectrometry (LC/MS).

AAV vector-mediated reversal of hypoglycemia in canine and murine glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.

ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease.

Tandem mass spectrometry was applied to detect derangements in the pathways of amino acid and fatty acid metabolism in N-ethyl-N-nitrosourea-treated (ENU-treated) mice. We identified mice with marked elevation of blood branched-chain amino acids (BCAAs), ketoaciduria, and clinical features resembling human maple syrup urine disease (MSUD), a severe genetic metabolic disorder caused by the deficiency of branched-chain alpha-keto acid dehydrogenase (BCKD) complex. However, the BCKD genes and enzyme activity were normal. Sequencing of branched-chain aminotransferase genes (Bcat) showed no mutation in the cytoplasmic isoform (Bcat-1) but revealed a homozygous splice site mutation in the mitochondrial isoform (Bcat-2). The mutation caused a deletion of exon 2, a marked decrease in Bcat-2 mRNA, and a deficiency in both BCAT-2 protein and its enzyme activity. Affected mice responded to a BCAA-restricted diet with amelioration of the clinical symptoms and normalization of the amino acid pattern. We conclude that BCAT-2 deficiency in the mouse can cause a disease that mimics human MSUD. These mice provide an important animal model for study of BCAA metabolism and its toxicity. Metabolomics-guided screening, coupled with ENU mutagenesis, is a powerful approach in uncovering novel enzyme deficiencies and recognizing important pathways of genetic metabolic disorders.