A novel functional assay for simultaneous determination of total fatty acid β-oxidation flux and acylcarnitine profiling in human skin fibroblasts using 2H31-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry
Introduction
Mitochondrial fatty acid β-oxidation (FAO) is the key energy generating process during fasting and in acute stress (e.g., infection) [1]. It also accounts for > 80% of energy supply to the heart and liver [2]. Inherited disorders of FAO have been extensively studied in the past 20 years due to the rapid advances in the understanding of these diseases and increasing numbers of diagnostic tools and treatment/dietary regimens [3], [4], [5], [6], [7], [8]. More than 20 disorders in fatty acid transport and mitochondrial β-oxidation have been reported, all are inherited as autosomal recessive diseases. The clinical presentations of FAO disorders (FAOD) may vary from neonatal-onset form with severe metabolic acidosis, hypoketotic hypoglycemia and cardiomyopathy, to late or adult-onset forms with myopathy or cardiomyopathy [9]. Many types of FAOD were also found in the Asian population [10], [11]. On the other hand, the common mutation K304E in medium-chain acyl-CoA dehydrogenase gene makes the disease a common FAOD in the Caucasian population [12]. As private mutations are frequent in other FAOD, mutation analysis of genes of the FAO pathway has not been used widely as the first-line laboratory investigation for these diseases. Instead, measurement of blood free carnitine and acylcarnitine profiling are used for screening of FAOD. Complementary to these blood tests, in-vitro studies of FAO flux and acylcarnitine profiling in cultured cells have been used in many specialized centers for definitive diagnosis of FAOD [4], [5], [7], [13].
Traditionally, the total FAO rate/flux is determined by monitoring the rate of production of CO2 in the form of 14CO2, or water in the form of 3H2O by cultured cells after incubation with either forms of radiolabeled fatty acids [14], [15], [16], [17]. There are obvious advantages of using tritium-labeled over 14carbon-labeled substrates because of its availability at higher specific activities and lower cost. Furthermore, majority of the radiolabel is recovered as 3H2O in the FAD- and NAD-linked dehydrogenation steps of the β-oxidation spiral, and subsequent release during oxidation of acetyl-CoA in the Kreb's cycle. However, both methods are tedious to perform and they require handling of radioactive substrates. Despite this, these assays represent a mean to truly quantitate the functional FAO rate [13].
In-vitro probe assay by acylcarnitine profiling, has been widely used as a complementary method for detecting specific FAO defects in cultured cells. Cells are incubated with different substrates, such as unlabeled-, [U-13C]-, or [16-2H3]-palmitic acid, for 72 to 96 h. Accumulation of any disease-specific acylcarnitines due to blockage in the FAO spiral can be determined by tandem mass spectrometry (MS/MS) [18], [19], [20]. This method has gained wide acceptance in many diagnostic laboratories and is complementary to specific enzyme assays and mutation analysis for definitive diagnosis of FAOD.
Recently, an in-vivo oxidation assay of stable isotope-labeled fatty acids, 2H31-palmitate, has been validated for quantitation of whole body fatty acid oxidation rate [21], [22]. Participants were dosed with 2H31-palmitate and enrichment of deuterated water (2H2O) in urine was measured by isotope ratio mass spectrometry (IRMS). The 2H31-palmitate oxidation rates were calculated from the results of 2H2O enrichment in spot urine samples collected hourly. We hypothesize that it may be possible to apply the same principle for in-vitro cell culture study for quantitation of total FAO rate and to substitute the radioactive assay for quantitation of FAO rate.
This paper reports the validation study of our novel functional assay by using universal deuterium-labeled fatty acids (2H31-palmitate or 2H27-myristate) to study simultaneously the (i) rate of FAO by measurement of deuterated water enrichment using IRMS and (ii) acylcarnitine profiling using ESI/MS/MS in cultured fibroblasts.
Section snippets
Culture media and reagents
Dulbecco's Modified Eagle Medium without glucose (DMEM-no glucose) or with 4500 mg/l glucose (DMEM-hi glucose), l-glutamine, newborn calf serum, penicillin/streptomycin solution (10,000 U/ml penicillin/10,000 μg/ml streptomycin), phosphate-buffered saline (PBS) and trypsin–EDTA were used, Invitrogen-Gibco Inc (Carlsbad, CA). l-carnitine and defatted bovine serum albumin were from Sigma Chemical Co (St. Louis, MO). 2H31-Palmitic and 2H27-myristic acids (98 at.%) were from Cambridge Isotope
Analytical performance of 2H2O enrichment and acylcarnitines measured by IRMS and ESI/MS/MS
The analytical performance of the IRMS was validated by studying the within- and between-batch precisions of the 2H/1H ratio measurements. Both water and culture media showed that at the level of 150 ppm (2H/1H ratio or 2H1HO/1H2O), the within- and between-batch precisions (CV) were < 1% (SD < 1 ppm). Total FAO rate experiments using 2H31-palmitate as substrate were repeated three times over a period of one month in two control and one FAO deficient cell lines. The between-batch CV of 2H2O
Discussion
Two separate assays (acylcarnitine profiling and total FAO rate/flux studies) in cultured fibroblasts have been used as the key investigations for FAO defects in specialized laboratories for nearly 2 decades. The techniques of acylcarnitine profiling have evolved from the tedious radio-HPLC [28], GCMS [29] to the most convenient, sensitive and specific ESI/MS/MS methods [19], [20]. On the other hand, determination of overall FAO rate/flux still relies on the release of radioactive tritiated
Acknowledgments
We thank Mr Enzo Ranieri for the supply of stable isotope-labeled acylcarnitines, and Simon Fung and Eric Pang for technical assistance.
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Contributed equally to this work.