Analysis of Acylcarnitines

Millington DS, Roe CR, Maltby DA (1984) Application of high resolution fast atom bombardment and constant B/E ratio linked scanning to the identification and analysis of acylcarnitines in metabolic disease. Biomed Mass Spectrom 11 236-241... 

Millington DS, Norwood DL, Kodo N, Roe CR, Inoue F (1989) Application of fast atom bombardment with tandem mass spectrometry and liquid chromatography/mass spectrometry to the analysis of acylcarnitines in human urine, blood, and tissue. Anal Biochem 180 331-339... 

Chace DH, DiPerna JC, Mitchell BL, Sgroi B, Hofman LF, Naylor EW. Electrospray tandem mass spectrometry for analysis of acylcarnitines in dried postmortem blood specimens collected at autopsy from infants with unexplained cause of death. Clin Chem 2001 47 1166-82. 

Figure 55-3 shows a flow chart for the postmortem evaluation of sudden and unexpected death that is centered on the analysis of acylcarnitines in blood and bile spots. Blood and bile could be conveniently collected on a single filter paper card, one identical to those used for newborn screening, which can be shipped at room temperature once properly dried. Both specimens should be collected to detect patients who may have only mild blood abnormalities when carnitine is depleted (Figure 55-4). In cases with a higher level of suspicion, an effort should be made to collect and freeze a specimen of liver and a skin biopsy, which could be analyzed only if needed to reduce cell culture workload. ...

Urine is of limited value and is not recommended for analysis of acylcarnitines [3]. Free and total carnitine determinations in urine are also generally of little diagnostic value. 

The evolution of tandem mass spectrometry (MS) for the analysis of acylcarnitines of blood and fibroblasts has been critical in the identification of previously unrecognized inborn errors of L-isoleucine degradation, 2-methylbutyryl-CoA dehydrogenase [11, 12] and 2-methyl-3-hydroxybutyr-yl-CoA dehydrogenase deficiencies [13]. 

This in fact is what happened. Tandem MS has clearly been shown to be the only technology to screen for disorders of fatty acid oxidation and could also detect many disorders of organic acid metabolism. Tandem MS has the ability to detect both compound classes (amino acids and acylcarnitines) and after demonstrating that both classes could be prepared in the same manner, the MS/MS analysis of blood spots for newborn screening applications was born. 

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. 

Acylcarnitine analysis was first performed in urine specimens in the evaluation of patients with organic acidemias. However, because it was found that acylcarnitine analysis of plasma is more informative for the diagnosis of FAO disorders than analysis of urine specimens, plasma has become the preferred specimen [17]. It is only recently that it was shown that urine acylcarnitine analysis still has a role in the diagnostic evaluation of patients with organic acidurias but uninformative or borderline abnormal results of plasma acylcarnitine and urine organic acid analysis [18-21]. In our laboratory, sample preparation and analysis is identical to that of plasma once a urine aliquot has been prepared that is based on the creatinine concentration. 

Acylcarnitine analysis of dried blood or bile spots is very similar to the analysis of plasma. A small disk (diameter typically 5 mm or less) is punched out of the blood spot and the acylcarnitines extracted by the addition of methanol and known concentrations of isotopically labeled acylcarnitines, which function as internal standards. The extract is dried under a stream of nitrogen, and derivatized by the addition of either n-butanol HC1 or n-methanol HC1. The acylcarnitines are measured as their butyl or methyl esters by MS-MS. The concentrations of the analytes are established by computerized comparison of ion intensities of these analytes to that of the internal standards. 

As noted for acylcarnitine analysis of other specimen types, the interpretation of results for blood and bile spots is based on quantitative reference ranges and ultimately pattern recognition by a trained biochemical geneticist. The reference ranges used in our laboratory for postmortem blood and bile spots represent prospectively updated percentile ranks (Table 3.2.4). 

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. 

Rashed MS, Ozand PT, Bennett MJ, Barnard JJ, Govindaraju DR, Rinaldo P (1995) Inborn errors of metabolism diagnosed in sudden death cases by acylcarnitine analysis of postmortem bile. Clin Chem 41 1109-1114... 

Costa CG, Struys EA, Bootsma A, et al (1997) Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography. J Lipid Res 38 173-182... 

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).

Diagnosis of the disorders discussed above requires the analysis of amino acids, organic acids, and acylcarnitines. Further details of such analytical. procedures are available on this book s accompanying Evolve site, found at http //evolve.elsevier.com/Tietz/textbook/. Here, we briefly describe the assay principles. 

Chace DH, Hillman SL, Vanhove JLK, Naylor EW. Rapid diagnosis of MCAD deficiency—quantitative analysis of octanoylcarnitine and other acylcarnitines in newborn blood spots by tandem mass spectrometry. Clin Chem 1997 43 2106-13. 

A free-standing liquid junction interface was coupled to a flat edge glass CE microchip for the analysis of small molecules (drugs, metabolites). It was demonstrated for the detection of recovered carnitine, acylcarnitines, imipramine, and desipramine spiked into urine or plasma at 5-500 p,g/mL level. Separations were typically performed in < 1 min and intra-assay precisions ranged from 4.1% to 7.3% Relative Standard Deviation RSD. A similar device, but fabricated in polymeric Zeonor material, was demonstrated for the analysis of carnitine standards. ... 

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.

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