Improved Stable Isotope Dilution-Gas Chromatography-Mass Spectrometry Method for Serum or Plasma Free 3-Hydroxy-Fatty Acids and Its Utility for the Study of Disorders of Mitochondrial Fatty Acid β-Oxidation

Abstract

Disorders of fatty acid oxidation (FAO) are difficult to diagnose, primarily because in many of the FAO disorders measurable biochemical intermediates accumulate in body fluids only during acute illness. Increased concentrations of 3-hydroxy-fatty acids (3-OH-FAs) in the blood are indicative of FAO disorders of the long- and short-chain 3-hydroxy-acyl-CoA dehydrogenases, LCHAD and SCHAD. We describe a serum/plasma assay for the measurement of 3-OH-FAs with carbon chain lengths from C(6) to C(16). We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay. The assay was linear from 0.2 to 50 micromol/L for all six 3-OH-FAs. CVs were 5-15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in micromol/L were as follows: 3-OH-C(6), 0.8 (0.3-2.2); 3-OH-C(8), 0.4 (0.2-1.0); 3-OH-C(10), 0.3 (0.2-0.6); 3-OH-C(12), 0.3 (0.2-0.6); 3-OH-C(14), 0.2 (0.0-0.4); and 3-OH-C(16), 0.2 (0.0-0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C(14) and 3-OH-C(16) concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C(14) to 3-OH-C(16). Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.