Elsevier

Journal of Chromatography B

Volume 823, Issue 2, 5 September 2005, Pages 122-130
Journal of Chromatography B

Enzymatic diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by detecting 2-octenoyl-CoA production using high-performance liquid chromatography: A practical confirmatory test for tandem mass spectrometry newborn screening in Japan

https://doi.org/10.1016/j.jchromb.2005.06.043Get rights and content

Abstract

Many of the previously described enzymatic assay methods for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency have been dependent upon the measurement of radioisotope-labeled co-products or reduction of electron acceptors. We have developed a direct assay method to detect 2-enoyl-CoA production using high-performance liquid chromatography (HPLC). Crude cell lysate prepared from lymphocytes were incubated with n-octanoyl-CoA and ferrocenium hexafluorophosphate. The detection of 2-octenoyl-CoA was significantly reproducible. We applied the assay to samples from four infants suspected to have MCAD deficiency by tandem mass spectrometry (MS/MS) newborn screening conducted in the Hiroshima area of Japan. Three of them were proved to have pathologically reduced residual enzyme activities, although they were associated with various clinical and biochemical phenotypes. In addition, another symptomatic Japanese patient and her presymptomatic sibling who were detected by MS/MS selective screening were successfully diagnosed by our enzymatic assay. These results indicate that the method can be a useful confirmatory test for MS/MS screening of MCAD deficiency.

Introduction

Tandem mass spectrometry (MS/MS) has enabled screening of inborn errors of fatty acid oxidation, including medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) deficiency. In order to aid the timely follow-up of screening results that suggest abnormalities in MCAD, rapid and simple confirmatory tests for the enzyme activity and/or gene mutation analysis should be available. For the enzymatic study, the assay which uses the reduction of electron transfer flavoprotein (ETF) has been utilized as a standard method [1], [2], [3]. However, since this method requires purification of pig liver ETF and must be performed under strictly anaerobic conditions, it is disadvantageous and other artificial electron acceptors, such as ferrocenium ion [3], [4], and phenazine methosulfate (PMS) in combination with dichlorophenol indophenol (DCIP) [5], have been utilized. The assay for tritium release from [2,3-3H]acyl-CoA was described as a radioisotope-dependent method [6]. In addition, intact-cell oxidation assays using the 14CO2 release [1], [4], [5] and the tritium release from [9,10-3H]fatty acids [7], [8], [9] were frequently utilized in previous studies.

Despite being the main product of the enzymatic reaction in the assays mentioned above, 2-enoyl-CoA production is not directly detected in these methods. There were several reports on the product formation in such assays. Kølvraa et al. detected the production of 3-hydroxyfatty acid using gas chromatography mass spectrometry (GC/MS) by coupling 2-enoyl-CoA production with crotonation and alkaline hydrolysis [10], and this method was utilized in other studies [11], [12]. Wanders [13] and Oey [14] referred to their method that detects the enoyl-CoA species produced by dehydrogenation of 3-phenylpropionyl-CoA using high-performance liquid chromatography (HPLC); however, the details of the method have not been described. In order to realize a practical method for enzymatic diagnosis of MCAD deficiency, we have developed another HPLC-based assay wherein n-octanoyl-CoA is used as substrate and 2-octenoyl-CoA production is directly detected. In this report, we will also demonstrate the application of our method to confirming MCAD deficiency in patients found through an MS/MS newborn screening program conducted in Japan.

Section snippets

Reagents

n-Octanoyl-CoA (MW 893.7) and flavin adenine dinucleotide (FAD) were purchased from Sigma Chemical (St. Louis, MO, USA). Ferrocenium hexafluorophosphate (FcPF6) was purchased from Aldrich (St. Louis, MO, USA). Acyl-CoA oxidase (ACO) was purchased from Wako Pure Chemical Industries (Osaka, Japan). All the other chemicals used were of the highest purity commercially available.

Preparation of crude cell lysate

Human lymphocytes were isolated from venous blood samples. Informed consent was acquired prior to blood sampling.

MS/MS newborn screening in the Hiroshima area

We have conducted a pilot study of MS/MS newborn screening in the Hiroshima area of Japan since 1999. All newborns in the area whose parents gave written informed consent in advance were enrolled in this study. Generally, dried blood spots (DBS) were collected on the fifth day after birth, and were analyzed by MS/MS. Details of the MS/MS protocol were described elsewhere [18]. Newborns showing elevated levels of octanoylcarnitine (C8-carnitine) in DBS (cut off < 0.3 nmol/ml) associated with the

Case reports

Patient 1 was a healthy 8-month-old boy of Japanese–Peruvian–European descent born in 2000, who abruptly became ill and rapidly fell into cardiopulmonary arrest associated with liver dysfunction and hyperammonemia (AST 286 IU/l, ALT 257 IU/l, CK 8702 IU/l, NH3 > 400 μg/dl). Plasma glucose at the onset was not measured. Fortunately, resuscitation and intensive care were started early, so that he recovered without any sequelae. The concentration of C8-carnitine and the C8/C10 ratio in serum collected

Results

Kinetic studies on the MCAD activity were initially performed using crude cell lysate prepared from 106 lymphocytes. Product formation linearly increased within the range of n-octanoyl-CoA concentration from 0.5 to 4 mmol/l (n = 4, Fig. 2a). Product formation with regard to FcPF6 concentration and incubation time exhibited an increase with a tendency to reach a plateau within the range tested; 0.25–4 mmol/l of FcPF6 (n = 3, Fig. 2b), and 5–30 min of incubation (n = 3, Fig. 2c). Adding FAD to the

Discussion

The introduction of acylcarnitine analysis by MS/MS has enabled mass screening of fatty acid oxidation disorders including MCAD deficiency. As the application of this technique to newborn screening prevails, there will be increasing need for simpler tests to confirm the abnormal results indicative of MCAD deficiency, especially in cases that do not have the common 985A > G mutation in the MCAD gene. Thus, it is worth establishing a rapid and simple method of enzymatic diagnosis. Various methods

Acknowledgements

The authors are grateful to Dr. Akira Ohtake, Department of Pediatrics, Saitama Medical School, Moroyama, Saitama, Japan, for offering blood samples of a symptomatic patient and her family. We are also grateful to Ms. Miyako Togawa, Ms. Junko Yanagawa, Ms. Miho Kubota, Ms. Chiyoko Yoshii, and Mr. Mochiyuki Hamakawa, Hiroshima City Clinical Research Center, Hiroshima, Japan, for their assistance in conducting the MS/MS newborn screening program. We express our gratitude to the Research Center

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