Quantitation of long-chain acylcarnitines by HPLC/fluorescence detection: application to plasma and tissue specimens from patients with carnitine palmitoyltransferase-II deficiency

Background: Carnitine palmitoyltransferase-II deficiency (CPT-II deficiency) is a rare disorder of lipid metabolism, in which the accumulation of long-chain acylcarnitines is a diagnostic marker. HPLC with fluorescence detection is an attractive analysis method due to its favorable combination of sensitivity, specificity, ease of analysis and minimal capital equipment costs. Methods: Long-chain acylcarnitines were isolated from tissue homogenates (0.5-2 mg wet weight) or plasma (50 μl) using silica gel columns and derivatized with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. Quantitation was by HPLC and fluorescence detection with standard curves (0.0-5.0 nmol/ml) for myristoyl-, palmitoleoyl-, palmitoyl-, oleoyl- and stearoylcarnitine using heptadecanoylcarnitine as the internal standard. Results: Significantly greater amounts of long-chain acylcarnitines were quantified in patients with CPT-II deficiency when compared to controls; e.g. (nmol/ml in patient plasma, controls mean±standard deviation): myristoylcarnitine (0.3, not detectable), palmitoleoylcarnitine (0.5, 0.1±0.1), palmitoylcarnitine (0.9, 0.1±0.0), oleoylcarnitine (3.0, 0.2±0.1), stearoylcarnitine (0.4, not detectable). Conclusions: This method can be used to quantitate long-chain acylcarnitines, illustrating their accumulation in CPT-II deficiency. The analysis was accomplished using inexpensive and widely available instrumentation and is appropriate for research investigators who require precise quantitation of long-chain acylcarnitines in complex biological samples.