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Tandem mass spectrometric determination of malonylcarnitine: diagnosis and neonatal screening of malonyl-CoA decarboxylase deficiency.

Malonic aciduria (OMIM 248360) is a rarely diagnosed autosomal-recessive inborn error of metabolism caused by congenital deficiency of malonyl-CoA decarboxylase (MCD; EC Hypoglycemia, seizures, developmental delay, and cardiomyopathy are among the most common symptoms, but both clinical signs and the time of presentation of patients with MCD deficiency can be variable. To date, only eight cases have been reported in the literature (1-8), and recently molecular defects within the MCD gene (MLYCD) have been elucidated for the first time in some of these cases (8-11). At least in part, clinical heterogeneity might be caused by the fact that MCD is expressed in different compartments of the cell and that MLYCD mutations with different effects on the subcellular localization of the MCD protein may thus affect different metabolic pathways (Wightman et al., submitted for publication).

To date, only symptomatic patients with MCD deficiency have been detected, and many of them were already severely handicapped at the time of diagnosis because of residues of acute metabolic crises or from episodes of cardiac decompensation, which may develop as a consequence of secondary carnitine deficiency. Typically, the key to diagnosis is the excessive amount of malonic acid in the patient's urine, which can be detected by gas chromatography-mass spectrometry. This then leads to a confirmatory test, such as the measurement of MCD activity in cell extracts, or to molecular genetic testing.

Detection of the carnitine ester of malonic acid has been mentioned previously in single cases of malonic aciduria (4, 6, 7). In the study reported here, we systematically investigated the concentration of malonylcarnitine in the blood of MCD-deficient patients by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Because MCD deficiency may be amenable to dietary and medical treatment, we also evaluated whether an increase in blood malonylcarnitine is detectable in the neonatal period, which would be mandatory for early presymptomatic diagnosis.

Details of the method used by our group have been reported previously (12). In summary, acylcarnitines were analyzed by ESI-MS/MS with an API 365 ESIMS/MS system equipped with a TurboIon spray device, Series 200 lp HPLC pump, and a Series 200 autosampler (PE-SCIEX). For that purpose, 3.2-mm (1/8-inch) spots were punched from Guthrie test cards by an automated punching device and transferred to a 96-well microtiter plate. After the addition of internal standard solution (containing 7.5 pmol of L- [[sup.2][H.sub.3]]octanoylcarnitine), methanol was added, and after elution, the methanolic extract was evaporated to dryness. Acylcarnitines were derivatized to the corresponding butyl esters with butanolic hydrochloric acid. Samples were redissolved in acetonitrile-water-formic acid (50:50:0.025 by volume) and introduced into the ionization chamber. Acylcarnitines were measured in multiple-reaction monitoring (MRM) mode with the ion pairs 347.4/85.0 Da and 360.4/85.0 Da for L-[[sup.2][H.sub.3]]octanoylcarnitine and malonylcarnitine, respectively. Malonylcarnitine was quantified relative to L-[[sup.2][H.sub.3]]octanoylcarnitine, assuming the same extraction and ionization efficacy for both carnitine esters.

Dried blood spots were obtained from two siblings with MCD deficiency at different time points. A detailed description of the clinical and biochemical data and the metabolic effect of their genotype (MLYCD 634insA/ 59insC) is to be published elsewhere (Wightman et al., submitted for publication). During the period of the study, both patients were in stable clinical condition without any signs of metabolic derangement. At the time of the first determination of malonylcarnitine, patient 1 was 4 years and 2 months of age, and at that time she was already on oral carnitine therapy; patient 2 was investigated for the first time at the age of 7 months before any treatment had been initiated. In addition, the neonatal Guthrie test cards of both patients were traced back, and the concentrations of acylcarnitines were also determined in these samples. To determine the reference interval for malonylcarnitine in dried blood samples, we analyzed 4333 unselected samples of healthy newborns.

At the time of the first determination, the blood malonylcarnitine concentration was 2.4 [micro]mol/L in patient 1 and 3.6 [micro]mol/L in patient 2. At the follow-up investigations, the malonylcarnitine concentration ranged between 1.66 and 3.78 [micro]mol/L (n = 8) in both patients. For comparison, in dried blood samples from the healthy controls, malonylcarnitine ranged from "not detectable" to 0.30 [micro]mol/L (99.5th centile; n = 4333). In the neonatal test card of patient 1, which had been stored for 4 years and 4 months, malonylcarnitine was not detectable. In contrast, in the test card of patient 2, stored for 9 months, the malonylcarnitine concentration was 0.40 [micro]mol/L.

The linearity and precision of the assay and the recovery of malonylcarnitine could not be calculated because malonylcarnitine is not commercially available. The detection limit (signal + 3 SD of a sample free of analyte) for malonylcarnitine was 0.08 [micro]mol/L, and the quantification limit (signal + 6 SD of a sample free of analyte) was 0.13 [micro]mol/L. To determine the stability of malonylcarnitine in dried blood samples stored at room temperature, we reanalyzed eight samples from patients with MCD deficiency after various storage times at room temperature. All samples showed a decrease in malonylcarnitine concentration. Regression analysis with curve fitting revealed significant agreement (P <0.001) with a logarithmic model (y = [e.sup.b*t] x 100%). In this equation, y is the concentration of malonylcarnitine relative to the first measurement, t is the time of storage between the first and the consecutive measurement, and the constant b was calculated to be -0.0028. With this model, the half-life ([t.sub.1/2) of malonylcarnitine in dried blood on filter paper stored at room temperature could be calculated to be 248 days (Fig. 1).

With this equation and the malonylcarnitine concentration measured in the screening card of patient 2 after 296 days of storage, the initial malonylcarnitine concentration was calculated to be 0.92 [micro]mol/L. With a presumptive malonylcarnitine concentration of ~1 [micro]mol/L in patients with MCD deficiency during the first days of life, the decrease in malonylcarnitine can be calculated. Concentrations should be expected to drop below the quantification limit after ~730 days. Even with a 10-fold higher initial concentration (10 [micro]mol/L), malonylcarnitine would no longer be detectable after ~1500 days. These considerations make it plausible that we could not detect malonylcarnitine in the screening card of patient 1 (after storage for 1514 days).


The diagnostic specificity of the test cannot yet be finally evaluated, although our preliminary data from 4333 newborns and the two patients with MCD deficiency seem to show that ESI-MS/MS has a high specificity for the detection of MCD deficiency. The highest concentration of malonylcarnitine in the healthy newborns was 0.43 [micro]mol/L, whereas the malonylcarnitine concentrations in dried blood samples from the two patients with MCD deficiency was 3.9- to 8.8-fold higher than the highest value of the reference population.

Several groups have reported that MCD-deficient patients benefit from a low-fat, high-carbohydrate diet, with the effects being improved urinary organic acid excretion and the avoidance of hypoglycemic episodes (2,3). Both our patients and cases from the literature (4) also showed a dramatic effect of carnitine treatment, which demonstrates that cardiac decompensation can be avoided in MCD-deficient patients. Finally, by analogy with fatty acid oxidation disorders such as medium-chain acyl-CoA dehydrogenase deficiency (13), one may conclude that knowledge of the diagnosis will improve outcome because early intervention is then possible in catabolic episodes that otherwise may lead to metabolic derangement. It therefore seems reasonable to suppose that early diagnosis and treatment can reduce the morbidity and mortality associated with MCD deficiency and to include MCD deficiency in neonatal metabolic screening programs. In this study, the malonylcarnitine concentration in the neonatal screening test card was increased in only one of the two patients investigated, but this was a problem of stability after sample storage for several years.

We conclude that (a) malonylcarnitine is detectable by ESI-MS/MS, (b) malonylcarnitine is increased in the blood of patients with malonic aciduria, and (c) ESI-MS/MS-based neonatal screening programs should be able to detect patients with MCD deficiency, a treatable metabolic disorder, before the development of symptoms.


(1.) Brown GK, Scholem RD, Bankier A, Danks, DM. Malonyl coenzyme A decarboxylase deficiency. J Inherit Metab Dis 1984;7:21-6.

(2.) Haan EA, Scholem RD, Croll HB, Brown GK. Malonyl coenzyme A decarboxylase deficiency. Clinical and biochemical findings in a second child with a more severe enzyme defect. Eur J Pediatr 1986;144:567-70.

(3.) MacPhee GB, Logan RW, Mitchell JS, Howells DW, Tsotsis E, Thorburn DR. Malonyl coenzyme A decarboxylase deficiency. Arch Dis Child 1993;69: 433-6.

(4.) Matalon R, Michaels K, Kaul R, Whitman V, Rodriguez-Novo J, Goodman S, et al. Malonic aciduria and cardiomyopathy. J Inherit Metab Dis 1993;16: 571-3.

(5.) Krawinkel MB, Oldigs HD, Santer R, Lehnert W, Wendel U, Schaub J. Association of malonyl-CoA decarboxylase deficiency and heterozygote state for haemoglobin C disease. J Inherit Metab Dis 1994;17:636-7.

(6.) Yano S, Sweetman L, Thorburn DR, Mofidi S, Williams JC. A new case of malonyl coenzyme A decarboxylase deficiency presenting with cardiomyopathy. Eur J Pediatr 1997;156:382-3.

(7.) Henderson MJ, Evans CE, Kumar V, Pourfazam M. Malonic aciduria presenting with developmental delay, malonyl carnitine increased in bloodspots [Abstract]. J Inherit Metab Dis 1998;21(Suppl 2):53.

(8.) Gao J, Waber L, Bennett MJ, Gibson KM, Cohen JC. Cloning and mutational analysis of human malonyl-coenzyme A decarboxylase. J Lipid Res 1999; 40:178-82.

(9.) Sacksteder KA, Morrell JC, Wanders RJ, Matalon R, Gould SJ. MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency. J Biol Chem 1999;274: 24461-8.

(10.) FitzPatrick DR, Hill A, Tolmie JL, Thorburn DR, Christodoulou J. The molecular basis of malonyl-CoA decarboxylase deficiency. Am J Hum Genet 1999;65:318-26.

(11.) Surendran S, Sacksteder KA, Gould SJ, Coldwell JG, Rady PL, Tyring SK, et al. Malonyl CoA decarboxylase deficiency: C to T transition in intron 2 of the MCD gene. J Neurosci Res 2001;65:591-4.

(12.) Rolinski B, Arnecke R, Dame T, Kreischer J, Olgembller B, Wolf E, et al. The biochemical metabolite screen in the Munich ENU mouse mutagenesis project: determination of amino acids and acylcarnitines by tandem mass spectrometry. Mamm Genome 2000;11:547-51.

(13.) Pourfarzam M, Morris A, Appleton M, Craft A, Bartlett K. Neonatal screening for medium-chain acyl-CoA dehydrogenase deficiency. Lancet 2001;358: 1063-4.

Rene Santer, [l*] Ralph Fingerhut, [2] Uta Ldssker, [l] Patrick J. Wightman, [3] David R. Fitzpatrick, [3] Bernhard Olgemdller, [2] and Adelbert A. Roscher, [4]

[1] Department of Pediatrics, University of Kiel, D-24105 Kiel, Germany; [2] Labor Becker, Olgemoller & Kollegen, D-81737 Munich, Germany; [3] MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom; [4] Dr. von Hauner Children's Hospital, Department of Clinical Chemistry and Biochemical Genetics, University of Munich, D-80337 Munich, Germany; * address correspondence to this author at: Department of Pediatrics, University of Kiel, Schwanenweg 20, D-24105 Kiel, Germany; fax 49-431-597-1831, e-mail santer@pediatrics.
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Title Annotation:Technical Briefs
Author:Santer, Rene; Fingerhut, Ralph; Lassker, Uta; Wightman, Patrick J.; Fitzpatrick, David R.; Olgemolle
Publication:Clinical Chemistry
Date:Apr 1, 2003
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