Evaluation of Metabolic Defects in Fatty Acid Oxidation Using Peripheral Blood Mononuclear Cells Loaded with Deuterium-Labeled Fatty Acids

Abstract

Because tandem mass spectrometry- (MS/MS-) based newborn screening identifies many suspicious cases of fatty acid oxidation and carnitine cycle disorders, a simple, noninvasive test is required to confirm the diagnosis. We have developed a novel method to evaluate the metabolic defects in peripheral blood mononuclear cells loaded with deuterium-labeled fatty acids directly using the ratios of acylcarnitines determined by flow injection MS/MS. We have identified diagnostic indices for the disorders as follows: decreased ratios of d₂₇-C14-acylcarnitine/d₃₁-C16-acylcarnitine and d₂₃-C12-acylcarnitine/d₃₁-C16-acylcarnitine for carnitine palmitoyltransferase-II (CPT-II) deficiency, decreased ratios of d₂₃-C12-acylcarnitine/d₂₇-C14-acylcarnitine for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, and increased ratios of d₂₉-C16-OH-acylcarnitine/d₃₁-C16-acylcarnitine for trifunctional protein (TFP) deficiency, together with increased ratios of d₇-C4-acylcarnitine/d₃₁-C16-acylcarnitine for carnitine palmitoyltransferase-I deficiency. The decreased ratios of d₁-acetylcarnitine/d₃₁-C16-acylcarnitine could be indicative of β-oxidation ability in patients with CPT-II, VLCAD, and TFP deficiencies. Overall, our data showed that the present method was valuable for establishing a rapid diagnosis of fatty acid oxidation disorders and carnitine cycle disorders and for complementing gene analysis because our diagnostic indices may overcome the weaknesses of conventional enzyme activity measurements using fibroblasts or mononuclear cells with assumedly uncertain viability.

Figure 1

Precursor ion mass spectra observed in acylcarnitine analysis by MS/MS. Homogenized PBMCs loaded with d₃₁-hexadecanoic acid were subjected to acylcarnitine analysis. (a) Healthy control, (b) CPT-II deficiency, and (c) VLCAD deficiency. ^∗Stable isotope-labeled acylcarnitines as internal standards.

Figure 2

Comparison of diagnostic ratios between healthy controls and patients with CPT-II deficiency, VLCAD deficiency, and TFP deficiency. (a) d₂₇-C14/d₃₁C16, (b) d₂₃-C12/d₂₇-C14, and (c) d₂₃-C12/d₃₁-C16. ^∗ p < 0.001, significant difference between the control and each disorder. The horizontal lines in controls represent the medians and interquartile ranges (25th–75th percentile).

Figure 3

Comparison of diagnostic ratios of d₂₉-C16-OH/d₃₁-C16 between healthy controls and patients with CPT-II deficiency, VLCAD deficiency, and TFP deficiency. ^∗ p < 0.001, significant difference between control and TFP deficiency. The horizontal lines in controls represent the medians and interquartile ranges (25th–75th percentile).

Figure 4

Comparison of diagnostic ratios and indexes between controls and patients with CPT-I deficiency. (a) d₇-C4/d₃₁-C16 after loading of d₃₁-hexadecanoic acid alone (p = 0.38). (b) d₇-C4/d₃₁-C16 after loading of d₁₅-octanoic acid and d₃₁-hexadecanoic acid (^∗ p = 0.012). (c) The adjusted ratio of d₇-C4/d₃₁-C16, corresponding to (b)/(a) (^∗ p = 0.012).

Figure 5

Comparison of the β-oxidation ability index (the ratios of d₁-C2/d₃₁-C16) between healthy controls and CPT-II deficiency, VLCAD deficiency, and TFP deficiency. ^∗ p < 0.001, significant difference between the control and each disorder. The horizontal lines in controls represent the medians and interquartile ranges (25th–75th percentile).

Figure 6

Correlation between the ratios of d₁-C2/d₃₁-C16 and the diagnostic ratios. Correlations between d₁-C₂/d₃₁-C16 and d₂₇-C14/d₃₁-C16 (a) and between d₁-C₂/d₃₁-C16 and d₂₃-C12/d₃₁-C16 (b) in CPT-II deficiency. Correlations between d₁-C2/d₃₁-C16 and d₂₇-C14/d₃₁-C16 (c) and between d₁-C2/d₃₁-C16 and d₂₃-C12/d₂₇-C14 (d) in VLCAD deficiency. The Spearman's rank correlation coefficient ρ and p values are indicated.