An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry
Introduction
Before activated long-chain fatty acids can be catabolized by the mitochondrial β-oxidation system, they must be first transported into the mitochondrial matrix. Since acyl-CoAs cannot cross the inner mitochondrial membrane, acyl-CoAs are first converted into the corresponding acyl-carnitine ester by Carnitine Palmitoyl Transferase I (EC 2.3.1.21, CPTI)1, an integral mitochondrial outer membrane protein. Subsequently, the acyl-carnitines are transported across the mitochondrial inner membrane by the carnitine/acyl-carnitine transporter (CACT). Finally, the acyl-carnitines are reconverted into their CoA-esters by Carnitine Palmitoyl Transferase II (CPTII) and the acyl-CoAs can enter the β-oxidation pathway [1], [2].
In liver, the activity of CPTI largely controls the flux through the mitochondrial β-oxidation pathway [3]. Hepatic CPTI activity is regulated by the concentration of malonyl-CoA, the first metabolite of fatty acid synthesis, which is produced from acetyl-CoA by the enzyme acetyl-CoA carboxylase. When glucose is plentiful, malonyl-CoA is produced and used for lipogenesis. The newly formed fatty acids are protected from oxidation through inhibition of CPTI by the high level of malonyl-CoA. Inhibition of CPTI, and therefore β-oxidation, is relieved when glucose levels fall and the hepatic malonyl-CoA concentration decreases [4].
Three genetically distinct isoforms of CPTI have been identified [5], [6], [7], [8], [9]. CPTI-a is the main isoform in liver, kidney, lung, spleen, intestine, pancreas, ovary, lymphocytes, and fibroblasts. CPTI-b is predominantly expressed in skeletal muscle, heart, adipose tissue, and testis. In brain both CPTI-a and CPTI-c are found. Low levels of the latter isoform are also detected in intestine, ovary and, testis [1], [9], [10], [11], [12].
To date, only deficiencies of the CPTI-a isoform have been described. Patients typically present in infancy with Reye-like attacks with hypoketotic hypoglycemia, hepatomegaly, elevated liver enzymes and hyperammonemia [13]. CPTI-deficient patients usually have elevated levels of free carnitine accompanied by the absence of medium- and long-chain acyl-carnitines in plasma and blood [14], [15]. Definite diagnosis of CPTI deficiency requires enzyme analysis followed by sequence analysis of the CPTI-a gene. Patient’s fibroblasts usually show 0–20% of control CPTI activity [13].
To date, one mass spectrometric method [16] and several radiometric methods [1], [11], [17], [18] have been described to measure CPTI activity. Unfortunately, these methods are rather laborious; samples are repeatedly extracted with butanol to separate the product, palmitoyl-carnitine, from the substrate, palmitoyl-CoA.
Here we report a novel, fast tandem-mass spectrometric (MS) assay without elaborate extraction procedures. Furthermore, we show that in most of the previously described methods CPTI activity has been underestimated and that inhibition of the mitochondrial β-oxidation pathway is essential for reliable and accurate measurement of CPTI activity.
Section snippets
Chemicals
[U-13C]-Palmitate was purchased from Advance Research Chemicals, CoASH from Roche, digitonin from Boehringer Mannheim. Carnitine, malonyl-CoA and d/l-octanoyl-carnitine were purchased from Sigma. d-Decanoyl-carnitine was a kind gift from the late J.D. McGarry (University of Texas Southwestern Medical Center, USA). The [2H3]-C3, [2H3]-C8 and [2H3]-C16-acyl-carnitines internal standards were obtained from Dr. Herman J. ten Brink (VU Medical Hospital, The Netherlands). [U-13C]-palmitoyl-CoA was
Development of the CPTI assay
The CPTI activity assays described to date are all rather laborious and usually involve the use of radio labeled substrate [1], [11], [16], [17], [18]. Because the product of CPTI, i.e., palmitoyl-carnitine, can be easily measured using tandem–MS [23], we investigated if we could develop a non-radioactive tandem-MS-based CPTI activity assay without elaborate extraction procedures.
In our assay we used intact fibroblasts, rather than sonicated cell homogenates as used by others [11], [16], and
Discussion
Here we describe the development of a fast and easy tandem-MS based CPTI activity assay. When we compared our new method with a radiometric and the other mass spectrometric CPTI activity assay [16], [18] we found a 4-fold higher CPTI activity with our new method. The lower activity measured in the other methods is possibly due to enzymatic degradation of the palmitoyl-carnitine produced during incubation. When this degradation is not inhibited, the CPTI activity is underestimated considerably.
Acknowledgment
The authors thank our mass spectrometry section for technical assistance.
References (26)
- et al.
Molecular enzymology of carnitine transfer and transport
Biochim. Biophys. Acta
(2001) Control of mitochondrial beta-oxidation flux
Prog. Lipid Res.
(2002)- et al.
Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA
J. Biol. Chem.
(1978) - et al.
Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function
J. Biol. Chem.
(1993) - et al.
High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone
FEBS Lett.
(1995) - et al.
Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I
Biochim. Biophys. Acta
(1996) - et al.
A novel brain-expressed protein related to carnitine palmitoyltransferase I
Genomics
(2002) - et al.
Expression of a cDNA isolated from rat brown adipose tissue and heart identifies the product as the muscle isoform of carnitine palmitoyltransferase I (M-CPT I). M-CPT I is the predominant CPT I isoform expressed in both white (epididymal) and brown adipocytes
J. Biol. Chem.
(1996) - et al.
Carnitine palmitoyltransferases 1 and 2: biochemical, molecular and medical aspects
Mol. Aspects Med.
(2004) - et al.
Carnitine palmitoyltransferase I activity monitoring in fibroblasts and leukocytes using electrospray ionization mass spectrometry
Anal. Biochem.
(1998)
Carnitine In Intermediary Metabolism. The Biosynthesis Of Palmitylcarnitine By Cell Subfractions
J. Biol. Chem.
Studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts: application to mitochondrial encephalomyopathies
Biochim. Biophys. Acta
Measurement of protein using bicinchoninic acid
Anal. Biochem.
Cited by (20)
Organic Acidemias and Disorders of Fatty Acid Oxidation
2020, Emery and Rimoin’s Principles and Practice of Medical Genetics and Genomics: Metabolic DisordersSubstrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism
2013, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :Glycerol 50% was added to the enzyme fractions which were stored at − 80 °C. Carnitine acetyltransferase activity was determined using the method described by van Vlies and co-workers for CPT1 [24]. The standard assay mixture contained 150 mM potassium chloride, 25 mM Tris–HCl pH 7.4, 2 mM EDTA, 10 mM potassium phosphate buffer pH 7.4, 1 mg/mL bovine serum albumin (BSA) essentially fatty acid free, 500 μM l-carnitine and 25 μM of each acyl-CoA to a final volume of 150 μL.
Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: Acyl-CoA profiles in short-chain fatty acid oxidation defects
2012, Molecular Genetics and MetabolismCitation Excerpt :Methods have not been readily available for tissue acyl-CoA measurement. Most published methods involve HPLC separations and all previously published methods do not detect a full range of acyl-CoA species, mainly due to hydrophobicity differences between long-, medium- and short-chain acyl-CoA species [5–16]. We have developed an analytical approach to directly measure fatty acyl-CoA species of all chain lengths using flow-injection tandem mass spectrometry.
Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling
2010, Biochimica et Biophysica Acta - Molecular Basis of DiseaseInhibition of hepatic carnitine palmitoyl-transferase I (CPT IA) by valproyl-CoA as a possible mechanism of valproate-induced steatosis
2010, Biochemical PharmacologyCitation Excerpt :The mean values of the two independent experiments were used for calculation. The differential quantification of fatty acylcarnitines was performed using ESI-MS/MS as described previously [35]. The peak height ratio of the formed [U-13C]-C16-carnitine to the peak height of the internal standard [2H3]-C16-carnitine was determined using MassLynx NT software version 4.0 (Waters – Micromass, Manchester, UK).
Mitochondrial long chain fatty acid β-oxidation in man and mouse
2009, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids