Tiffany Thomas (Presenter)
Columbia University Medical Center
Bio: Tiffany Thomas is a associate professor at Columbia University Medical Center in the department of Pathology and Cell Biology. She is the director of the CUMC/NYP Biochemical Genetics Laboratory. She received her Ph.D. from the University of California, Berkeley in Molecular and Biochemical Nutrition and did her postdoctoral training at Columbia University with Henry Ginsberg studying lipid metabolism. Her research focuses on the metabolic profiles of patients with inherited and acquired diseases with the goal of discovering novel biomarkers for disease diagnoses as well as monitoring disease progression using high throughput/high sensitivity mass spectrometry based assays.
Authorship: Tiffany Thomas, Irina Genkin, Michael Pesce
Columbia University Medical Center, New York, NY
| Short Abstract Analysis of plasma acylcarnitine profiles plays an important role in the diagnosis and management of patients with inborn errors of fatty acid metabolism. A pattern of elevated long-chain acylcarnitines (C16 to C18:2-carnitine) is associated with impaired carnitine cycle function as well as defective long-chain fatty acid metabolism. Blood collected from 3 subjects was immediately processed (spun, plasma separated, frozen) or left at room temperature for 2, 4, 6, 8, 24, or 48 hours and subsequently processed and analyzed by FI-ESI-MS/MS. Specimens left unprocessed for greater than 8 hours were associated with elevated C16 to C18:2-carnitine in all three subjects. No elevation of long-chain acylcarnitines was observed in plasma left at room temperature. Careful control of pre-analytical handling time is imperative for the correct interpretation of plasma acylcarnitine profiles. |
Long Abstract
Carnitine is a naturally occurring amino acid derivative with a key role in fatty acid metabolism as well as in the detoxification of non-metabolizable products of intermediary metabolism resulting from organic acidopathies. Inborn errors of metabolism can result in rapid metabolic decompensation, coma, and ultimately death if not detected and treated quickly. Analysis of the plasma acylcarnitine profile plays an important role in the diagnosis of a host of fatty acid oxidation defects and organic acidurias. The correct interpretation of the acylcarnitine profile is critical for the diagnosis and subsequent treatment of patients with suspected inborn error of metabolism.
Patients with impaired long-chain fatty acid metabolism usually present with hypoglycemia without compensatory ketosis due to the inability to metabolize fatty acids to acetyl-CoA. The severity of the phenotype is variable depending on the affected enzyme and amount of residual enzyme activity, but range from early onset cardiomyopathy and multi-organ failure to a more mild myopathic form with recurrent myalgias and elevated serum creatine kinase (CK). Patients with an impairment in the carnitine cycle affecting fatty acid oxidation such as carnitine palmitoyltransferase II (CPT-II) deficiency and carnitine-acylcarnitine translocase (CACT) deficiency as well as patients with defective long-chain fatty acid metabolism (very long-chain acyl-CoA dehydrogenase deficiency; VLCAD) can present with elevated long-chain acylcarnitines due to impaired transport of acylcarntine across the mitochondrial inner-membrane into the mitochondrial matrix (CACT deficiency), trans-esterification of acylcarnitine to acyl-CoA in the mitochondrial matrix (CPT-II deficiency), or decreased function of the first enzyme in mitochondrial beta-oxidation (VLCAD deficiency). Elevation of long-chain acylcarnitines can be mild in these patients in between episodes of CKemia or if the specimen was collected after commencement of intravenous glucose, therefor mildly elevated long-chain acylcarntines in patients with clinical suspicion of inborn errors of metabolism is cause for concern.
In order to investigate the role of prolonged pre-analytical specimen handling time, blood from three healthy volunteers was collected in lithium heparin tubes. The tubes were gently inverted for 30 seconds then the blood was aliquoted into separate tubes that were immediately processed (spun, plasma separated into a new tube, frozen at -20C), or left at room temperature for 2, 4, 6, 8, 24, or 48 hours and subsequently processed to mimic hospital conditions by which a sample might accidentally be left on the bench for an extended period. Additionally, separate aliquots of plasma that was freshly processed were left at room temperature for 2, 4, 6, 8, 24 or 48 hours. After the end of the study, all samples for each subject were processed and analyzed together.
To 20ul of plasma, proteins were removed by precipitation with 500ul of methanol containing a mixture of isotope-labeled internal standards. Acylcarnitines were derivatizated into their butyl esters and subsequently quantified in multiple reaction monitoring (MRM) mode by tandem mass spectrometry (FI-ESI-MS/MS), using a product ion unique to acylcarnitines, m/z=85.
All three of the subjects had normal acylcarnitine profiles when analyzed from plasma that was immediately processed. Within 6 hours, the concentration of C16-, C18, C18:1, and C18:2 increased between 1.3X to 2.3X, 8 hours 1.7X to 4.7x, and by 48 hours 2.5X to 8.3X relative to the freshly processed sample. The acylcarnitine profiles for all 3 subject was abnormal after 8 hours at room temperature. No difference was observed in any of the long-chain acylcarntines for plasma left at room temperature relative to plasma that was immediately frozen consistent with the hypothesis that the elevation of long-chain acylcarnitines is from the dissociation of acylcarnitines from erthyrocytes. In conclusion, careful monitoring and control of the specimen processing time is imperative to ensure acylcarnitine profile artifacts from prolonged specimen handling time are avoided which may result in the incorrect diagnosis and treatment of the patient.
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