The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results

CPT1 catalyses the following reaction: Currently, only deficiencies of liver CPT1 have been reported. Fortunately, this CPT1 isoform is expressed in fibroblasts and lymphocytes. In principle, there are several ways to measure the activity of CPT1. A frequently used method involves incubation of freshly prepared tissue homogenates (Demaugre et al. 1988), isolated mitochondria, or selectively permeabilized cells (Schaefer et al. 1997) in a medium containing palmitoyl-CoA plus [¹⁴C]-labeled carnitine, followed by extraction of the [¹⁴C]-labeled acylcarnitine using water-saturated butanol at the end of the incubation period. Incubations are usually performed in the presence and absence of malonyl-CoA (>100 μmol/L) and the malonyl-CoA-inhibitable activity is taken to represent true CPT1-activity. We have been using this method to determine the activity of CPT1 in cultured skin fibroblasts using digitonin to permeabilize the plasma membrane for quite a few years. Recently, however, we came across some serious pitfalls of this assay, one being the fact that the acylcarnitine produced by CPT1 turned out to undergo appreciable further oxidation (van Vlies et al. 2007). The implication of this finding was that the activity of CPT1 had been underestimated considerably. We have studied this phenomenon in detail and have found out that this problem could simply be overcome by addition of KCN, an inhibitor of cytochrome C oxidase and thereby of the mitochondrial oxidative phosphorylation system. Addition of KCN blocked further oxidation of palmitoylcarnitine completely (van Vlies et al. 2007). With KCN present in the assay medium CPT1 activities as measured in fibroblasts were approximately two-fold higher as compared to assay conditions without KCN (van Vlies et al. 2007). It should be noted, however, that this finding does not mean that the previously used method was not suitable for diagnosing patients with deficiencies of CPT1. In order to avoid the use of radioactive chemicals, we have modified the assay and turned it into a highly sensitive, non-radioactive tandem-MS-based assay by using [U-¹³C]-palmitoyl-CoA rather than [1-¹⁴C]-palmitoyl-CoA. The assay was validated by measuring the activity of CPT1 in cell lines of five patients with a full, genetically confirmed deficiency of CPT1, which revealed no detectable CPT1 activity (van Vlies et al. 2007). We have been using this assay for a few years now and have found that it is a very robust and powerful method. Although it is not a standard procedure, the assay can also be applied to cell and tissue homogenates.

CACT catalyzes the transport of carnitine and acylcarnitines of various chain lengths by an exchange mechanism (Palmieri 2004). CACT activity can only be measured either in freshly prepared mitochondria, or in selectively permeabilized cells which is the method preferred by ourselves. Murthy et al. (1986) developed an assay for the carnitine/acylcarnitine translocase applicable to biopsied specimens without requiring isolation of mitochondria. This method was adapted by Pande et al. (1993) and modified to allow measurement of CACT-activity in permeabilized fibroblasts using digitonin as permeabilizing agent. This assay measures the formation of [¹⁴C]-acetylcarnitine from added [2-¹⁴C]-pyruvate in the mitochondrion and requires the active participation of the pyruvate transporter, pyruvate dehydrogenase, CRAT, and CACT to allow uptake of carnitine (needed in the CRAT reaction) in exchange for radiolabeled acetylcarnitine (Fig. 3a). Furthermore, malonate was added to the incubation medium to prevent degradation of acetyl-CoA in the Krebs cycle by inhibiting succinate dehydrogenase and thereby blocking the generation of oxaloacetate (Fig. 3a).

As an alternative to the use of radiolabeled pyruvate, which may be troublesome at times, we have developed a different method in which the formation of [¹⁴C]-CO₂ from [1-¹⁴C]-acetylcarnitine is measured to determine CACT activity (IJlst et al. 2001). The principle of our method is that the [1-¹⁴C]-acetylcarnitine enters the mitochondrion via CACT followed by the conversion of [1-¹⁴C]-acetylcarnitine into [1-¹⁴C]-acetyl-CoA catalyzed by CRAT after which the radiolabeled acetyl-CoA is oxidized in the Krebs cycle generating [¹⁴C]-CO₂ (Fig. 3b).

Although the two methods developed by Pande et al. (1993), Murthy et al. (1986) and ourselves (IJlst et al. 2001) work very well in practice, a drawback of the two methods is that both assays are not true single-enzyme (or in this case single-transporter) assays since both methods require the active participation of a number of additional auxiliary enzymes (Fig. 3). There is definitely a need for a single one-step assay.

Since the reaction catalyzed by CPT2, i.e. palmitoylcarnitine + CoASH → palmitoyl-CoA + carnitine, is readily reversible, the activity of CPT2 can be measured either in the forward or backward reaction. The notion that long-chain acylcarnitines can easily be extracted from aqueous solutions by means of butanol has led to the development of a very popular assay in which tissue homogenates, mitochondria, or membrane preparations are incubated in a medium containing radiolabeled carnitine and palmitoyl-CoA plus a detergent like Triton X-100 to inactivate CPT1, followed by extraction of radiolabeled palmitoylcarnitine. Although this method works fine, alternative assays have been developed in which CPT2 is measured in the forward, physiological direction like we do in our laboratory (see Table 2). An important reason for this is that the maximal rate of CPT2 in the forward reaction is markedly higher than in the reverse direction. In fibroblasts from CPT2-deficient patients with either deletions or mutations in the CPT2 gene leading to premature stopcondons, the activity of CPT2 is fully deficient using the assay developed in our laboratory.

Several alternative assays have been described in the literature, including the one described by Rettinger et al. (2002). In this elegant and powerful assay, a coupled reaction system is used in which the carnitine produced from palmitoylcarnitine in the CPT2 reaction is directly converted into acetylcarnitine, which is then quantified by tandem mass-spectrometry.

The specific measurement of each of the individual ACADs in a mitochondrial preparation, or preferably in total cell homogenates, requires at least two things: (1) a specific substrate preferably reactive with only one particular ACAD; and (2) a specific detection system allowing the activity of each ACAD to be measured. The first condition is only met for MCAD if 3-phenylpropionyl-CoA is used as substrate since 3-phenylpropionyl-CoA is a unique substrate for MCAD (Lehman et al. 1990). Although palmitoyl-CoA is a substrate for multiple ACADs (see Fig. 4a), we and others have found that palmitoyl-CoA is a specific substrate for VLCAD in homogenates of cultured skin fibroblasts and lymphocytes. This is concluded, among others, from the observation that the acyl-CoA dehydrogenase activity measured with palmitoyl-CoA as substrate is fully deficient in fibroblasts (Fig. 5a) and in lymphocytes (Fig. 5b) from patients with genetically confirmed VLCAD-deficiency (Fig. 5a). This indicates that ACAD9 does not contribute to the palmitoyl-CoA dehydrogenase activities in these cell types despite its significant expression levels. For SCAD, a specific substrate which is not handled by any of the other ACADs has not yet been identified. Butyryl-CoA is usually used as substrate. Since butyryl-CoA is also a substrate for other ACADs (Fig. 4a), corrections have to be made for the contribution by the other ACADs. One method which we favor in our laboratory is to measure the butyryl-CoA dehydrogenase activity in homogenates before and after immuno-precipitation of SCAD using a specific antibody (Wanders et al., in preparation).

In the early years, acyl-CoA dehydrogenase activities were measured fluorimetrically and/or spectrophotometrically using different dye-reduction assays, which are based on the use of artificial electron acceptors such as dichlorophenol-indophenol (DCPIP) with either phenazine methosulphate (PMS) or ETF (Rhead et al. 1983) as mediator. These types of assays have a number of drawbacks, including the relatively high background plus the fact that only isolated mitochondria or even more purified fractions can be used in the assay. Although different improved variations on this theme have been devised (Dommes and Kunau 1976), there was a clear need for new assays. One was the ETF-based assay, which has remained the method of choice for many years for measurement of SCAD, MCAD, and VLCAD in patients’ cells (Stanley et al. 1983; Coates et al. 1985). Oxidized ETF is highly fluorescent, and this fluorescence is lost upon the reduction of ETF by any of the acyl-CoA dehydrogenases, thus yielding a sensitive and accurate technique. Unfortunately, ETF has never become commercially available and, in addition, the assay needs to be done anaerobically, which in practice yields problems. For this reason alternative assays have been devised including the tritium-release assay for MCAD using [2,3-³H]-octanoyl-CoA as substrate (Amendt and Rhead 1985).

Kolvraa et al. (1982) were the first to device a completely different method based on the identification of the products of the ACAD reaction, in their case MCAD, using gas chromatography-mass spectrometery. Different variations on this theme have been published by several authors (Niezen-Koning et al. 1992, 1994; Duran et al. 1992).

In 1990, Lehman and Thorpe (1990) published a direct spectrophotometric method for MCAD based on the use of ferricenium hexafluorophosphate. The method involves measurement of the 3-phenylpropionyl-CoA mediated reduction of the ferricenium ion at 303 nm. We have adopted the same principle to measure the activity of VLCAD (IJlst et al. 1994). However, it soon turned out that these spectrophotometric methods for MCAD and VLCAD, respectively, lack robustness and sensitivity, and, more importantly, lead to artificially high residual activities, amounting to 50% of normal in fibroblasts from patients with a full deficiency of MCAD (Taylor et al. 1992). Similar high residual activities were found for VLCAD in fibroblasts using the ferricenium-based assay in fibroblasts from patients with a full deficiency of VLCAD (IJlst and Wanders 1993a).

Since ferricenium hexafluorophosphate was (and is) commercially available, we (Wanders et al. 1999; Oey et al. 2006) devised a new type of assay for measurement of ACAD activities, which involves the use of ferricenium hexafluorophosphate as electron acceptor followed by the analysis of the products of the ACAD reaction by HPLC coupled to UV-detection or by UPLC coupled to tandem-MS detection (Table 2). This method is now also used by others (Spiekerkoetter et al. 2003; Tajima et al. 2005, 2008; ter Veld et al. 2009). We have been using this assay for VLCAD, MCAD, and SCAD analysis (see Table 3). Moreover, we are using the same methodology for the analysis of other ACADs, which include glutaryl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, short branched-chain acyl-CoA dehydrogenase, and ACAD8.

Figure 6 shows typical HPLC/UV chromatograms of our MCAD assay for fibroblasts (Fig. 6a) and lymphocytes (Fig. 6b) from control subjects and MCAD-deficient patients homozygous for the c.985A>G mutation. The finding of a virtually full deficiency of MCAD in both fibroblasts and lymphocytes of patients homozygous for the 985A>G mutation (Fig. 6) allows easy discrimination between controls and patients and inspired us to propose direct analysis of MCAD in lymphocytes in neonates with a positive neonatal screening result for MCAD, as a first-line test next to acylcarnitine analysis in a new blood sample. Figure 7 shows our results obtained in 2007 and 2008, the first two years of the Dutch extended neonatal screening program. The results for the 16 neonates with a positive screening result for MCADD in 2007 show that 11 of the 16 neonates analyzed by us were clearly MCAD-deficient. In the remaining 5 neonates, MCAD activities were not deficient, with activities ranging between 40 and 180% (Fig. 7a). In 2008, 10 of the 15 neonates suspected to have MCADD were clearly MCAD-deficient, whereas a fully normal activity was found in neonate 15. Four out of the 15 neonates had intermediate values for MCAD in lymphocytes ranging from 21% in neonate 11 to 39% in neonate 14 (Fig. 7b). Neonates 13 and 14 turned out to be true heterozygotes for MCADD. Detailed follow-up studies in fibroblasts from neonates 11 and 12, however, revealed that both had true MCAD-deficiency, albeit mild. This was concluded from the results of the palmitate loading test in which cells are loaded with palmitate followed by acylcarnitine profiling (Ventura et al. 1999), which revealed an abnormal profile with a mildly elevated octanoylcarnitine level. Furthermore, measurement of MCAD-activity in fibroblasts from neonates 11 and 12 revealed clear deficiencies. Molecular analysis revealed mutations in both MCAD alleles in these patients. A full account of these data will be published separately.

The same strategy is used for the follow-up of neonates with a positive VLCAD screening result. Figure 8 shows the results obtained in 2007 and 2008 with respect to neonates with an abnormal acylcarnitine profile pointing to VLCAD deficiency as detected upon neonatal screening. In 2007, only one neonate was identified in the Netherlands with a positive screening result suggestive for VLCADD. We received blood from this neonate, isolated lymphocytes, and found a partial deficiency pointing to mild VLCAD-deficiency (Fig. 8). In 2008, three neonates were picked up by neonatal screening with an acylcarnitine profile suggestive for VLCAD. In two of these neonates, VLCAD was measured in lymphocytes with clear deficiencies in both of them (Fig. 8).

The activity of the different 3HADs is usually measured in the reverse direction because the equilibrium of the 3HAD-reaction drives the reaction backwards, unless the product of the 3HAD-reaction is constantly metabolized via the direct and continuous thiolytic cleavage of the 3-ketoacyl-CoA ester, the product of the 3HAD-reaction. 3HAD activities can simply be measured by following the 3-ketoacyl-CoA ester-dependent consumption of NADH at 340 nm. Fortunately, the activity of SCHAD can be measured directly in skin fibroblast and lymphocyte homogenates using acetoacetyl-CoA as substrate. Acetoacetyl-CoA is unique for SCHAD, because the LCHAD component of MTP is fully unreactive towards this substrate (Fig. 4c). Indeed, in fibroblasts of genetically proven SCHAD-deficient patients, the 3HAD-activity as measured with acetoacetyl-CoA was almost fully deficient, as shown in Fig. 9a for the two established SCHAD-deficient patients (siblings) identified by Molven et al. (2004) with intermediate activities in fibroblasts from the parents. A similar marked deficiency of SCHAD was found in lymphocytes from the only patient with proven SCHAD-deficiency we have analyzed so far (Fig. 9b).

Unfortunately, a specific substrate only reacting with the LCHAD-component of MTP has not yet been identified. In our hands, 3-ketopalmitoyl-CoA is the best substrate for LCHAD at present. In fibroblasts, about 75% of the activity as measured with 3-ketopalmitoyl-CoA is catalyzed by LCHAD, whereas the remaining 25% is derived from SCHAD. We have searched for methods allowing the assay to be more specific. One method involves the selective immunoprecipitation of SCHAD from homogenates prior to enzymatic analysis using antibodies raised against SCHAD. Although the method works fine in practice, we prefer the second method we devised which is based on the finding that N-ethylmaleimide, a SH-reagent, is a powerful inhibitor of LCHAD with no effect on SCHAD (IJlst and Wanders 1993b). In practice, this means that we measure the 3-ketopalmitoyl-CoA dehydrogenase activity in the absence and presence of N-ethylmaleimide. The difference between the two rates then represents true LCHAD-activity. Figure 10a, b shows the results of such LCHAD-activity measurements in fibroblasts (Fig. 10a) and lymphocytes (Fig. 10b) from 8 and 5 genetically proven LCHADD-patients, respectively.

The most popular assay for measurement of KAT activities is that in which the consumption of the 3-ketoacyl-CoA ester is measured spectrophotometrically at 303 nm. The method is based on the notion that 3-ketoacyl-CoAs absorb at 303 nm, when magnesium ions are present. As discussed before, mitochondria contain at least three different thiolases of which the short-chain thiolase with 2-methylacetoacetyl-CoA as specific substrate is inactive in the absence of potassium ions. Furthermore, MCKAT is fully inactive with 3-ketopalmitoyl-CoA (see Fig. 4d) so that the thiolase activity of MTP can be measured specifically by using 3-ketopalmitoyl-CoA as substrate in a medium lacking potassium ions.

Although ETF-alpha, ETF-beta, and ETF-dehydrogenase are strictly speaking not directly involved in fatty acid beta-oxidation, they do play an essential role by transferring the electrons coming from the acyl-CoA dehydrogenases to the respiratory chain at the level of coenzyme Q. Measurement of ETF-alpha, ETF-beta, and ETF-dehydrogenase has notoriously been difficult. In order to measure ETF-dehydrogenase, for instance, purified ETF is required. In our laboratory, we favor direct sequence analysis of the ETFA, ETFB, and ETFDH genes in any patients suspected to suffer from glutaric aciduria type 2 based on urinary organic acid analysis and/or acylcarnitine analysis. Sequence analysis of these genes is available in our laboratory. It should be noted that direct enzymatic analysis of ETF and ETF-DH is operational in other laboratories including the Lyon-lab, headed by Christine Vianey-Saban (personal communication).

Since acylcarnitine profiling in plasma from patients may be so stunningly predictive in terms of which enzyme or transporter is deficient (Fig. 2), whole cell fatty acid oxidation studies have lost its importance as a first line test. Rather, fatty acid oxidation studies are only done if direct enzymatic analysis has not led to the identification of the enzyme defect. Furthermore, whole cell fatty acid oxidation studies are warranted to determine the effect of a certain deficiency of an enzyme on flux through the mitochondrial beta-oxidation system. This is especially relevant in case of partial enzyme deficiencies as found in patients picked up by neonatal screening programs as described above from MCADD (Fig. 7).

Through the years, many different methods have been set up for this purpose, including: (1) [¹⁴C]-CO₂ release assays using different [1-¹⁴C]- or [U-¹⁴C]-labelled fatty acids; (2) tritium release assays using different [³H]-labelled fatty acids, notably [9,10-³H]-myristic acid, [9,10-³H]-palmitic acid, and [9,10-³H]-oleic acid (Manning et al. 1990; Olpin et al. 1992, 1997); and (3) quantitative acyl-CoA and acylcarnitine profiling studies. The latter methods have been pioneered by Bartlett and co-workers in the late 1980s during studies on the beta-oxidation of fatty acids in rat liver mitochondria. The methodology involved incubation of mitochondria with radiolabeled fatty acids followed by extraction of the acyl-CoA esters followed by separation on radio-HPLC with continuous on-line monitoring of radioactively labeled acyl-CoAs (Watmough et al. 1988, 1989).

These methods were later improved and extended to include the analysis of both acyl-CoA esters and acylcarnitines, and were subsequently used to study mitochondrial fatty acid oxidation in fibroblasts from patients first using mitochondria isolated from fibroblasts (Kler et al. 1991) and later using selectively permeabilized fibroblasts (Pourfarzam et al. 1994) and peripheral blood cells (Schaefer et al. 1995).

In 1995, Roe and co-workers (Nada et al. 1995) published an elegant method named the acylcarnitine profiling method which involves incubation of intact fibroblasts or lymphoblastoid cells with a deuterated long-chain fatty acid ([17,17,18,18-²H₄]-linoleic acid) followed by analysis of the acylcarnitines using tandem-mass spectrometry. The profiles are often very characteristic which immediately guides the way to the underlying defect. Several variations on this theme have been published by different groups the main difference being the use of differentially labelled fatty acids including [16-²H₃]-palmitic acid (Vianey-Saban et al. 1998), [U-¹³C]-palmitic acid (Ventura et al. 1999), and [15,15,16,16,16-²H₅]-palmitic acid (Sim et al. 2002; Law et al. 2007).

We have been using the acylcarnitine profiling method with [U-¹³C] palmitic acid as substrate (Ventura et al. 1999) and the tritium-release assays using tritiated oleic acid for many years now. The reason for subjecting each patient cell line to both these assays is that the tritium-release assay measures the actual rate of fatty acid oxidation with no information on the site of the enzyme block whereas the reverse is true for the acylcarnitine profiling methods, which do provide information on the site of the enzymatic block. Recently, Law et al. (2007) devised an elegant method which couples acylcarnitine profiling via ESI/MS/MS with the simultaneous measurement of the flux through the beta-oxidation pathway, using [²H₃₁]-palmitic acid. The rate of fatty acid oxidation was measured from the deuterated water enrichment by means of isotope ratio MS.

It should be emphasized that there is still room for improvement of the currently available whole cell assays since patients with a mild defect in mitochondrial fatty acid oxidation may show no abnormalities in either of the two tests (results not shown). We have experienced this through the years especially in patients with mild deficiencies at the level of VLCAD and LCHAD/MTP, as well as in mild glutaric aciduria type 2 patients. We are continuing our efforts to improve these methods.