Acylcarnitine analysis

Acylcarnitine analysis for the diagnosis of organic acidemias and particularly of FAO disorders plays an increasingly prominent role in all venues of clinical biochemical genetics prenatal diagnosis, newborn screening, evaluation of symptomatic patients, and postmortem screening. Almost exclusively performed by tandem... 

Acylcarnitine analysis using stable-isotope-labeled internal standards provides quantitative data for acylcarnitine species [14]. However, to provide meaningful results to referring healthcare providers, it is critical to complement analytical proficiency with in-depth interpretation of results, as is true for many other examples of complex metabolic profiles [39]. 

Fig. 3.2.1 Structures of carnitine, acylcarnitine and butylated acylcarnitine. The R represents the acylcarnitine species with up to 18 carbons, which are typically the aim of an acylcarnitine analysis...

Table 3.2.1 Clinically relevant acylcarnitine species included in a typical acylcarnitine analysis and their relevance when abnormally elevated (unless otherwise noted). BKT fi-ketothiolase,...

This chapter focuses on acylcarnitine analysis in various sample types following derivatization to butyl- or methylesters and flow injection ESI-MS/MS. Capillary electrophoresis MS/MS and LC-MS/MS methods have also been described in recent years with the noted benefits of isomer separation and further simplification of the sample preparation steps, although at the cost of larger sample volume requirements and longer analytical times [53, 54]. 

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. 

Fig. 3.2.4 Structures of the isomers 2-methylbutyryl- and isovalerylcarnitine. These isomers can not be differentiated by acylcarnitine analysis as described here because both appear as butylated Cs-acylcarnitine esters at m/z 302. Differentiation would require an additional chromatography step prior to tandem mass spectrometry analysis...

Another antibiotic that may cause problems in the interpretation of butylated acylcarnitines is cefotaxime (Fig. 3.2.5d) [63]. This antibiotic, or metabolites thereof, reveals itself by acylcarnitine analysis at m/z 470, which is otherwise considered to represent the monounsaturated form of 3-hydroxy hexadecenoylcarnitine (Ci i-OH). In poorly resolved scans this may be difficult to differentiate from m/z 472, which is a marker for LCHAD and TFP deficiencies. However, whereas m/z 472 (C16-OH) is more abundant than C16 1-OH in these FAO disorders, the profile of a patient treated with cefotaxime usually reveals an m/z 470 to m/z 472 ratio that is greater than 1. Furthermore, and in contrast to cefotaxime treatment, both LCHAD and TFP deficiencies are usually accompanied by elevations of other long-chain species (Table 3.2.1) [57]. 

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. 

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 in Dried Blood and Bile Spots by MS/MS... 

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. 

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. 

After 72 h incubation at 37°C, 1.0 ml of medium is removed for acylcarnitine analysis. It can be spotted on filter paper immediately or stored frozen for later acylcarnitine analysis. The remaining medium is discarded. Then, 2.0 ml of 0.85% sterile saline solution is added to each well and each flask gently washed. The saline solution is then replaced by 0.2 ml of 0.25% Trypsin-EDTA solution. After 3-5 min the cells will lift off the plate bottom, which should be confirmed by fight microscopy. To bring the cells back into suspension, 1.0 ml Dulbecco s 1 x PBS (Gibco Labo-... 

Acylcarnitine analysis is performed as precursor scan in positive ion mode. Q1 is set to scan a mass range from m/z 200 to 500 m/z, while Q3 is set to determine a precur-... 

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... 

Retrieve newborn screening card for blood spot acylcarnitine analysis... 

I Blood and bile spots on filter paper for acylcarnitine analysis Frozen liver for Oil Red O stain, metabolic profile and carnitine I Fibroblast culture for in vitro enzymatic and molecular studies... 

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).

Van Hove JL, Zhang W, Kahler SG, Roe CR, Chen YT, Terada N, et al. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency diagnosis by acylcarnitine analysis in blood. Am J Hum Genet 1993 52 958-66. 

---