Plasma acylcarnitine

A variety of body fluids can be used for acylcarnitine analysis. While initially the favored specimen, urine acylcarnitine analysis is the least appropriate when an FAO disorder is under diagnostic consideration. Heparinized plasma or whole blood spotted on filter paper are preferred in this context. 

A quality control (QC) standard mixture, a normal control and an abnormal control are run with each batch. The normal control is made by aliquoting 100-pl portions of a normal range pooled plasma into screw-cap vials. The abnormal control is made by spiking 50 ml of normal pooled plasma with defined concentrations of unlabeled acylcarnitine standards spanning the mass range covered by the analysis (see Reagents and Chemicals). The concentration for each standard should be chosen based on the upper limit of the reference range for the respective acylcarnitine species. The... 

Table 3.2.2 Reference ranges for plasma acylcarnitine species (pmol/l) in different age groups...

Fig. 3.2.2 Profiles of acylcarnitines as their butyl esters in plasma (precursor of m/z 85 scan) of a normal control (a), a patient with glutaric acidemia type I and low carnitine levels (b), and a patient with glutaric acidemia type I and normal carnitine status (c). Peak 1 free carnitine (m/z 218), peak 2 acetylcarnitine (C2 m/z 260), peak 3 glutarylcarnitine (C5-DC m/z 388). The asterisks represent the internal standards (from left to right) d3-acetylcarnitine (C2 m/z 263), d3-propionylcarnitine (C3 m/z 277), d3-butyrylcarnitine (C4 m/z 291), d3-octanoylcarnitine (C8 m/z 347), d3-dodecanoylcarnitine (Ci2 m/z 403), and d3-palmitoylcarnitine (Ci6 m/z 459)...

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. 

Fig. 3.2.5 Profiles of acylcarnitines as their butyl esters in plasma (precursor of m/z 85 scan) of a normal control (a) and patients with various organic acidemias. Propionylcarnitine (C> m/z 274 peak 3) is the primary marker for both propionic acidemia (b) and methylmalonic acidemias (c). Note that an elevation of methylmalonylcarnitine (C4-UC m/z 374) is not typically found in patients with methylmalonic acidemias. In the three cases of ethylmalonic encephalopathy (d) analyzed in our laboratory, elevations of ,- (m/z 288 peak 4) and C5-acylcarnitine (m/z 302 peak 5) species were noted. Isolated C5-acylcarnitine elevations are encountered in patients with isovaleric acidemia (e), where it represents isovalerylcarnitine. Cs-Acylcarnitine is also elevated in patients with short/branched chain acyl-CoA dehydrogenase deficiency, where it represents 2-methylbutyrylcarnitine (see Fig. 3.2.4), and in patients treated with antibiotics that contain pivalic acid, where it represents pivaloylcarnitine [20, 59, 60]. Patients with /3-ketothio-lase deficiency (f) present with elevations of tiglylcarnitine (C5 i m/z 300 peak 6) and C5-OH acylcarnitine (m/z 318 peak 7). In most cases of 3-methylcrotonyl-CoA carboxylase deficiency (g) Cs-OH acylcarnitine is the only abnormal acylcarnitine species present. The differential diagnosis of C5-OH acylcarnitine elevations includes eight different conditions (Table 3.2.1). Also note that C5-OH acylcarnitine represents 3-hydroxy isovalerylcarnitine in 3-methylcrotonyl-CoA carboxylase deficiency (g), and 2-methyl 3-hydroxy butyrylcarnitine in / -ketothiolase deficiency...

The creatinine concentration is measured in the sample using routine methods (i.e., the Jaffe reaction). A urine volume equivalent to 0.25 mg creatinine is diluted to 300 pi with deionized water (if the creatinine equivalent exceeds 300 pi, no dilution is made). A 20-pl aliquot of the diluted or undiluted urine is then analyzed following the procedure described above for plasma acylcarnitine analysis (section 3.2.4). The final result is expressed as mmol/mol creatinine. 

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. 

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. 

The reagents used for filter paper analysis are the same as for plasma acylcarnitine analysis except for the internal standard/extraction solution, which is prepared using methanol as opposed to acetonitrile. 

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... 

Schmidt-Sommerfeld E, Penn D, Duran M, et al (1992) Detection and quantitation of acylcarnitines in plasma and blood spots from patients with inborn errors of fatty acid oxidation. Prog Clin Biol Res 375 355-362... 

Ghoshal AK, Guo T, Soukhova N, Soldin SJ (2005) Rapid measurement of plasma acylcarnitines by liquid chromatography-tandem mass spectrometry without derivatization. Clin Chim Acta 358 104-112... 

Van Hove JL, Kahler SG, Feezor MD, et al (2000) Acylcarnitines in plasma and blood spots of patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency. J Inherit Metab Dis 23 571-582... 

Laboratory studies Serum transaminases, blood glucose, plasma and urine carnitine and acylcarnitines, urinary organic acids... 

All enteral feeds were stopped. The baby was given intravenous glucose, L-arginine, sodium benzoate, and sodium phenylacetate. Hemodialysis was initiated. At this time, there were no spontaneous respirations, there was no response to painful stimuli, and brainstem reflexes were absent. The plasma amino acid results revealed a glutamine level of 1500 pmol/L (normal 254-823), and citrulline was undetectable (normal 10-34 pmol/L). Quantitative carnitine, plasma acylcarnitine, and urine organic acid profiles were normal. The urine orotic acid concen-... 

Diagnosis of mitochondrial myopathies is initially clinical, involving phenotypic (observable expression of characters and traits) evaluation, followed by laboratory evaluation. If an mtDNA mutation is detected, diagnosis is relatively straightforward in the absence of an mtDNA mutation, diagnosis becomes difficult. Laboratory studies may include blood plasma or cerebral spinal fluid measurement for lactic acid, ketone bodies, plasma acylcarnitines and organic acids in the urine. If they are abnormal, a muscle biopsy is performed. 

Figure 55-14 Plasma profiles of plasma acylcarnitine butyl-ester derivatives. A, Normal control. B, Propionic acidemia. C, Short-chain acyl-CoA dehydrogenase deficiency. D, Isovaleric acidemia. E, Medium-chain acyl-CoA dehydrogenase deficiency. F, Very long-chain acyl-CoA dehydrogenase deficiency. G, Long-chain L-3-hydroxy acyl-CoA dehydrogenase deficiency.The symbol marks internal standards [ Hjj-acetylcarnitine (m/z 263) [ HaJ-propionylcarnitine (m/z 277) fH ]-butyrylcarnitlne (m/z 295) pHal-octanoylcarnitine (m/z 347) [ Haj-dodecanoylcarnltine (m/z 403) [ Haj-palmitoy I carnitine (m/z 459).

C6-C14 3 hydroxydicarboxylic aciduria (Figure 55-11, F) with prominent unsaturated species, and elevated plasma concentrations of C16-OH and C18-OH acylcarnitine species (Figure 55-14, G). These metabolites could not be found in specimens collected when patients are clinically asymptomatic. 

Acylcarnitine reference intervals are listed for plasma in Table 55-6, for urine in Table 55-7, and for postmortem blood and bile in Table 55-8. The last two are calculated as the 5 to 95 percentile range of all postmortem cases previously investigated in our laboratory. 

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. ... 

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