Urine acylcarnitine analysis

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. 

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. 

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).

Urine samples for carnitine and acylcarnitine analysis should be frozen and shipped on dry ice (note however that urine is not recommended as a specimen for diagnosis, especially for defects of fatty acid oxidation). For convenience where distance from the testing laboratory is an issue, urine for these tests can also be spotted onto cotton fiber filter paper, allowed to dry and mailed in an envelope. Amniotic fluid should be frozen and shipped on dry ice. 

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... 

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. ... 

The laboratory diagnosis of inborn errors of metabolism cannot be completed by analysis of amino acids, organic acids, and acylcarnitines alone. Many defects are situated in other parts of the metabolic chart such as the breakdown of nucleotides and glycoproteins or the biosynthesis of cholesterol (and other steroids) or glycoproteins. Accumulating metabolites or biosynthetic intermediates may be analyzed in plasma or urine by a variety of techniques. The most widely used approaches are described here. 

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. 

Urine for organic acid analysis is collected at 0-3 h and 3-6 h after the load. Insufficient ketogenesis (less than twofold increase of the starting value) is observed in long-chain fatty acid oxidation defects and HMGS-defi-ciency. Controls have been shown to accumulate unsaturated C12-C16 acylcarnitines this profile is different from any of the primary defects [18]. Patients with any of the primary defects will accumulate the relevant acylcarnitines and (hydroxy) fatty acids in plasma. Defects distal from CPT2-defi-ciency are additionally characterized by the excretion of dicarboxylic acids in the urines after loading. 

---