Acylcarnitine profile

Figure 14.2. Acylcarnitine profile obtained using a precursor ion scan of 85 Da. The profile is from the blood spot of a normal patient.

Acylcarnitine profiles are dependent on the clinical status of the patient at the time of sample collection [56, 57]. It is therefore important to provide the biochemical genetics laboratory with information regarding the clinical context during which the sample was collected. The laboratory must be conscientious of the fact that carnitine deficiency states can be associated with acylcarnitine profiles that lack any acylcarnitine species that are elevated above the reference range. Therefore, it is essential that the complete profiles are reviewed and even borderline elevated acylcarnitines should prompt further follow up in the presence of abnormally low free acetylcar-nitine (Fig. 3.2.2). If clinically indicated, a repeat sample should be collected at least 24 h after L-carnitine supplementation. 

Formiminoglutamate (FIGLU), a marker for glutamate formimino-transferase deficiency, was recently also shown to be detectable by acylcarnitine analysis represented as a peak with m/z 287 (Fig. 3.2.3d) [64]. In poorly resolved acylcarnitine profiles, this peak may be confused with iso-/butyrylcarnitine (m/z 288). To avoid the incorrect interpretation of acylcarnitine profiles, we recommend performing the analysis in product scan mode as opposed to multiple reaction monitoring (MRM) mode. For example, the FIGLU peak at m/z 287 would not have been correctly identified in MRM mode because the transition of 287 to 85 is typically not selected. However, the 288/85 transition would reveal abnormal results, but in fact not represent either butyryl- or isobutyrylcarnitine, but another FIGLU related ion species. 

As is true for plasma acylcarnitine analysis, the interpretation of urine acylcarnitine profiles is based on quantitative reference ranges and pattern recognition. The reference ranges used in our laboratory for urine are provided in Table 3.2.3. 

Cell medium is pooled after incubation of several negative cell lines and added to the acylcarnitine analysis of each batch as controls. The mean and standard deviation is calculated with a minimum of ten between-run values. A standard solution made up of equimolar amounts of standard and internal standard solution will undergo acylcarnitine analysis with each batch (standard control). The expectation is that the standard and corresponding internal standard peaks are of equal abundance in the acylcarnitine profile and that no other acylcarnitine peaks are present. 

Millington DS, Kodo N, Norwood DL, Roe CR (1990) Tandem mass spectrometry a new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. J Inherit Metab Dis 13 321-324... 

Ventura FV, Costa CG, Struys EA, et al (1999) Quantitative acylcarnitine profiling in fibroblasts using U-C-13 palmitic acid an improved tool for the diagnosis of fatty acid oxidation defects. Clin Chim Acta281 l-17... 

Giak Sim K, Carpenter K, Hammond J, Christodoulou J, Wilcken (2002) Quantitative fibroblast acylcarnitine profiles in mitochondrial fatty acid beta-oxidation defects phenotype/me-tabolite correlations. Mol Genet Metab 76 327-334... 

Okun JG, Kolker S, Schulze A, et al (2002) A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency. Biochim Biophys Acta 1584 91-98... 

Young SP, Matern D, Gregersen N, et al (2003) A comparison of in vitro acylcarnitine profiling methods for the diagnosis of classical and variant short chain acyl-CoA dehydrogenase deficiency. Clin Chim Acta 337 103-113... 

Schulze-Bergkamen A, Okun JG, Spiekerkotter U, et al (2005) Quantitative acylcarnitine profiling in peripheral blood mononuclear cells using in vitro loading with palmitic and 2-oxoadipic acids biochemical confirmation of fatty acid oxidation and organic acid disorders. Pediatr Res 58 873-880... 

Roe DS, Yang BZ, Vianey-Saban C, Struys E, Sweetman L, Roe CR (2006) Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts. Mol Genet Metab 87 40-47... 

Rizzo C, Boenzi S, Wanders RJA, Duran M, Caruso U, Dionisi-Vici C (2003) Characteristic acylcarnitine profiles in inherited defects of peroxisome biogenesis a novel tool for screening diagnosis using tandem-MS. Pediatr Res 53 1-6... 

To put the discussion described above in a practical illustration, I have included a mass spectrum from a newborn blood spot of a patient confirmed to have medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency. Figure 8.1.3 is an acylcarnitine profile obtained from a methanol extract of a dried blood spot. Stable-isotope acylcarnitine internal standards were mixed with the methanol extracting solvent at a concentration that is equivalent to 1 or 2 pmol/1 of blood. The concentrations of each internal standard are marked on the illustration by the clear hexagons. 

Fig. 8.1.3 Acylcarnitine profile of a blood spot from a newborn with medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency...

Figure 55-4 Postmortem diagnosis of MCAD deficiency by acylcarnitine analysis of blood and bile collected at autopsy.The patient was a 3-year-old, previously healthy child who had symptoms of a viral respiratory tract infection. He was a compound heterozygote for the common 985A>G mutation and another mutation.The symbol marks the internal standards, same amount added to both specimens. A, Blood acylcarnitine profile.The concentrations of acetyicarnitine (C2), hexanoylcarnitine (C6), octanoylcarnltine (C8), and decenoylcarnitine (CIO I) were 2.8,0.3, 1.4,and 0.3pmol/L, respectively (for reference intervals see Table 55-8). B, Bile acylcarnitine profile (after lOx dilution).The concentrations of C2,C6,C8,and C10 i were 52.8, 73.1,665.6, and l8i.3pmo /L, respectively (for reference intervals see Table 55-8).The bile/biood C8 ratio was 475. In postmortem urine, hexanoylglycine was also markedly elevated (69.6mmoi/mol creatinine reference interval 0.1 to 1.3).

Rinaldo P, Cowan TM, Matern D. Acylcarnitine profile analysis. Genet Med. 2008 10(2) 151-6. 

Figure 3 Tandem mass spectra from a typical analysis of the derivatized extract of a newborn blood spot. The five panels represent five types of analyses In a single 2 min run. These Include (left to right, top to bottom), an analysis of free carnitine, short chain acyl earn nines, acylcarnitine profile, amino acid profile, basic amino acids. The stars represents internal standards for quantification.

The first and most widely used MS/MS test developed for clinical diagnosis is the acylcarnitine profile [3]. This chapter will therefore focus primarily on this test and its diagnostic implications. Other valuable tests have recently been developed or are in the process of development by MS/ MS, some of which are also discussed in this section. These include assays for free and total carnitine [17], selected amino acids including phenylalanine and tyrosine [4], methionine [5], homocysteine [6] and sulfocysteine, and for selected pyrimidines [7] and acylglycines [8]. Methods for bile acids [9, 10], steroids, plasmalogens [11], sphingolipids and phospholipids have also recently been reported. Although alternative methods are available for most of these tests, none can match the quantitative precision and rapid turn-around time of MS/MS. 

Table E.7. Artefacts and nonspecific abnormalities in plasma and blood acylcarnitine profiles...

In metabolic diseases, disease-specific acylcarnitines can accumulate and elevate the acyl/free carnitine ratio. However, a normal ratio is often seen in affected patients when under good metabolic control. Therefore, it is important to understand that neither the absolute concentrations nor the ratio of free and total carnitine values are predictive of metabolic disease. Therefore, if metabolic disease is suspected, it is recommended that the acylcarni-tine profile test be ordered as well as the free and total carnitine assay. The acylcarnitine profile is a separate test, designed to recognize defects of intermediary metabolism in which abnormal acyl-CoA metabolites accumulate in mitochondria and are exported to the plasma as acylcarnitines. 

Very low plasma total carnitine levels (i.e. <15 pM) with a normal acyl-carnitine pattern could signal any of a number of acquired deficiencies (diet or drug related) or a deficiency of the plasma membrane transporter. Patients with certain metabolic disorders, especially GA-I and fatty acid oxidation disorders such as MCAD and VLCAD, who have never received carnitine supplement can become markedly carnitine deficient, and their acylcarnitine profiles may be interpreted as normal if pathognomonic metabolite levels do not exceed the normal cut-off. Carnitine deficiency or in-... 

Another patient [12] was a 3-year-old product of a consanguineous mating who had hypotonia and retarded motor development. MRI was normal. He had 2-methylbutyrylglycinuria, but a normal acylcarnitine profile. His asymptomatic mother also excreted 2-methylbutyrylglycine. The activity of 2-methylbutyryl-CoA dehydrogenase in fibroblasts was 10% of control. Mutation analysis revealed homozygosity for a G>A 1228 transition in the patient and his mother. 

Amino acids Organic acids Carnitine AcylgLycines Plasma Amino acids Organic acids Carnitine Biotinidase Acylcarnitine profile CSF... 

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