An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry

https://doi.org/10.1016/j.ymgme.2006.07.006Get rights and content

Abstract

Carnitine palmitoyl transferase I (CPTI), which converts acyl-CoA and carnitine into acyl-carnitine and free CoASH, is the rate limiting enzyme of hepatic mitochondrial β-oxidation. CPTI-deficiency is a severe disorder characterized by Reye-like attacks with hypoketotic hypoglycemia, hepatomegaly, elevated liver enzymes and hyperammonemia. We developed a simple tandem-MS-based assay to measure CPTI activity in human fibroblasts. Surprisingly, a large part of the palmitoyl–carnitine formed in our assay by CPTI was degraded into C14- to C2-acyl-carnitines. Degradation of the product of CPTI leads to under estimation of the CPTI activity. When we used potassium cyanide to inhibit enzymes downstream of CPTI and thereby degradation of the product, we measured four times more CPTI activity than the previous methods. This inhibition is essential for correct calculation of CPTI activity. In fibroblasts of CPTI-deficient patients, CPTI activity was not detectable and this assay can be used for the diagnosis of CPTI-deficiency.

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)

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