©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for Intermediate Channeling in Mitochondrial -Oxidation (*)

(Received for publication, July 21, 1994; and in revised form, October 17, 1994)

Mohamed A. Nada (1)(§) William J. Rhead (2) Howard Sprecher (3) Horst Schulz (4) Charles R. Roe (1)

From the  (1)Department of Pediatrics, Division of Biochemical Genetics, Duke University Medical Center, Research Triangle Park, North Carolina 27709, the (2)Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242, the (3)Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210, and the (4)Department of Chemistry, City College of the City University of New York, New York, New York 10031

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The accumulation of beta-oxidation intermediates was studied by incubating normal and beta-oxidation enzyme-deficient human fibroblasts with [^2H(4)]linoleate and L-carnitine and analyzing the resultant acylcarnitines by tandem mass spectrometry. Labeled decenoyl-, octanoyl-, hexanoyl-, and butyrylcarnitines were the only intermediates observed with normal cells. Intermediates of longer chain length, corresponding to substrates for the beta-oxidation enzymes associated with the inner mitochondrial membrane, were not observed unless a cell line was deficient in one of these enzymes, such as very-long-chain acyl-CoA dehydrogenase, long-chain 3-hydroxyacyl-CoA dehydrogenase, or electron transfer flavoprotein dehydrogenase. Matrix enzyme deficiencies, such as medium- and short-chain acyl-CoA dehydrogenases, were characterized by elevated concentrations of intermediates corresponding to their respective substrates (octanoyl- and decenoylcarnitines in medium-chain acyl-CoA dehydrogenase deficiency and butyrylcarnitine in short-chain acyl-CoA dehydrogenase deficiency). These observations agree with the notion of intermediate channeling due to the organization of beta-oxidation enzymes in complexes. The only exception is the incomplete channeling from thiolase to acyl-CoA dehydrogenase in the matrix. This situation may be a consequence of only one 3-ketoacyl-CoA thiolase being unable to interact with the several acyl-CoA dehydrogenases in the matrix.


INTRODUCTION

Investigations of the control and organization of the enzymes involved in mitochondrial fatty acid beta-oxidation have been impeded by the inability to specifically identify and quantitate the steady-state concentrations of intermediates of the pathway in the unperturbed intact cell. Studies of fatty acid oxidation intermediates with subcellular fractions, such as isolated mitochondria, may be influenced by alterations affecting the structural organization and integrity of important interacting systems, such as the respiratory chain and other tightly integrated pathways of mitochondrial metabolism. The importance of identifying specific intermediates in deficient human cell lines is exemplified by the detection of tetradecadienoate in cells incubated with linoleate (Kelley, 1992). This observation suggested the existence of a very-long-chain acyl-CoA dehydrogenase. However, the complete spectrum of beta-oxidation intermediates is not easily resolved. Similarly, the rate of oxidation of radiolabeled fatty acids, such as [1-^14C]palmitate, [9,10-^3H(2)]palmitate, etc., can, at best, reveal a reduced rate of beta-oxidation without revealing fatty acid intermediates indicative of specific enzyme defects (Kolvraa et al., 1982; Saudubray et al., 1982; Moon and Rhead, 1987; Manning et al., 1990).

An interest in the control and organization of fatty acid oxidation in intact human cells, as well as a need to analyze the potential perturbations resulting from specific inherited enzyme deficiencies, prompted the development of a dynamic steady-state system in which all intermediates could be identified simultaneously by tandem mass spectrometry. Incubation of normal cells or cells with specific defects in fatty acid oxidation with [17,17,18,18-^2H(4)]linoleate ([^2H(4)]C) revealed intermediates reflecting the overlapping chain length specificities of the acyl-CoA dehydrogenases, unique profiles of intermediates associated with specific enzymatic defects, and information relevant to the organization of the enzymes of fatty acid beta-oxidation.


EXPERIMENTAL PROCEDURES

Materials

Acylcarnitine standards, L-carnitine, and defatted bovine serum albumin (BSA) (^1)were purchased from Sigma; the cell culture media, antibiotics, and fetal calf serum were obtained from Life Technologies, Inc. [15,16-^3H]Palmitate was purchased from Amersham Corp. [^2H(9)]Isovalerylcarnitine and [^2H(3)]palmitoylcarnitine were gifts of Sigma Tau Pharmaceutical Inc. (Rome, Italy). L-[^2H(9)]Carnitine was synthesized by demethylation and remethylation of carnitine with perdeutro methyliodide (C^2H(3)-I) as described previously (Millington et al., 1989). [^2H(9)]Octanoylcarnitine was synthesized as described (Bohmer and Bremer, 1958).

Synthesis of Stable Isotope-labeled Linoleic Acid

9,12-[17,17,18,18-^2H(4)]Octadecadienoic acid ([^2H(4)]linoleic acid) was synthesized in two steps. 1-[7,7,8,8-^2H(4)]Bromo-2-octyne was prepared by coupling 4-pentyn-1-ol with ethyl vinyl ether followed by reduction of the triple bond with deuterium gas in the presence of Wilkinson's catalyst (Rakoff and Rohwedder, 1992). The blocking group was cleaved, and 1-[4,4,5,5-^2H(4)]pentanol was reacted with HBr to give 1-[^2H(4)]bromopentane, which was converted to 1-[^2H(4)]bromo-2-octyne (Sprecher and Sankarappa, 1992). The product, 1-[^2H(4)]bromo-2-octyne, was coupled with the diGrignard adduct of 9-decynoic acid (Osbond et al., 1961) to yield 9,12-[17,17,18,18-^2H(4)]octadecadiynoic acid, which in turn was reduced to [17,17,18,18-^2H(4)]linoleic acid with Lindlar's catalyst in ethyl acetate. The product was homogeneous by gas chromatographic/mass spectrometric analysis as both methyl- and t-butyldimethylsilyl esters. The isotopic purity determined by mass spectrometry was 94.7% ^2H(4), 3.0% ^2H(3), and 2.1% ^2H(2).

Cell Culture and Preparations

Fibroblasts were cultured in Eagle's minimal essential medium containing nonessential amino acids, 10% fetal calf serum, and antibiotics. Cells were harvested by trypsinization and washed in Hanks' balanced salt solution. The cells were resuspended in bicarbonate-buffered Eagle's minimal essential medium with 10% (v/v) fetal calf serum before use in in vitro incubations. Studies were conducted using human skin fibroblasts from passages 6 to 20.

Substrate Preparation and Fatty Acid Oxidation Assay

Aliquots from stock solutions of [17,17,18,18-^2H(4)]linoleic acid in ethanol were prepared by completely evaporating the ethanol under a stream of dry N(2) and then dissolving the residue in a solution of defatted BSA in medium. The molar ratio of fatty acid to BSA was 4:1.

Skin fibroblasts (0.12-0.16 mg of protein) were subcultured in 75-cm^2 flasks and left to grow for 24 h, after which the medium was replaced with 5.0 ml of freshly prepared bicarbonate-buffered Eagle's minimal essential medium containing 10% fetal calf serum, 0.4 mML-carnitine, and 0.2 mM labeled fatty acid bound to BSA. Cells were incubated for the specified period of time at 37 °C in humidified 5% CO(2), 95% air. After the incubation period, the reaction mixture was decanted, and the cells were trypsinized. The cells and media were then recombined.

To 0.1-ml aliquots of the aqueous cell suspension were added 3 pmol of [^2H(9)]isovalerylcarnitine ([^2H(9)]C(5)), 20 pmol of [^2H(9)]octanoylcarnitine ([^2H(9)]C(8)), and 40 pmol of [^2H(3)]palmitoylcarnitine ([^2H(3)]C) as internal standards. The concentrations of acylcarnitines corresponding to chain lengths of C(4), C(5), and C(6) were determined based on their intensities relative to the internal standard [^2H(9)]C(5). The concentrations of C(8) and C were measured relative to the concentration of the internal standard [^2H(9)]C(8). Those of C(14)-C(18) were determined based on their relative concentrations to [^2H(3)]C. The mixture was extracted with 0.8 ml of methanol and centrifuged, and the clear supernatant was dried under nitrogen and immediately prepared for analysis of acylcarnitines. Data in Table 1were generated from six different incubations of the same normal cell line and three different incubations of each of the defective cell lines used in this study. The profiles shown in Fig. 1are a representative obtained from three different cell lines for each defect and at least three different incubations for each cell line. Protein concentrations were determined by the method of Bradford(1976) with BSA as a standard.




Figure 1: Acylcarnitine profiles of normal and fatty acid oxidation-deficient human cultured fibroblasts incubated (for 96 h) with L-carnitine and [^2H(4)]linoleic acid. The signals represent the molecular ions of acylcarnitine butyl esters detected by tandem MS. [^2H(9)]Isovalerylcarnitine, [^2H(9)]octanoylcarnitine, and [^2H(3)]palmitoyl-carnitine were added to each culture as internal standards (i.s.; m/z 311, 353, and 459, respectively). Intermediates detected are hydroxyoctadecadienoyl (C18:2(OH))-, hydroxyhexadecadienoyl (C16:2(OH))-, octadecadienoyl (C18:2)-, hexadecadienoyl (C16:2)-, tetradecadienoyl (C14:2)-, decenoyl (C10:1)-, octanoyl (C8)-, hexanoyl (C6)-, butyryl (C4)-, and acetylcarnitines (C2). Asterisks denote labeled intermediates.



Oxidation rates of [^3H(2)]palmitate were determined as described (Moon and Rhead, 1987), except that the concentration of [15,16-^3H]palmitate was 80 µM. Fibroblast cultures from five normal controls and each patient were assayed in duplicate three to five times.

Sample Preparation and Instrumentation

The dried aliquots containing acylcarnitines were incubated with 100 µL of 3 M HCl in 1-butanol at 65 °C for 15 min in a capped 1-ml glass vial. The esterifying agent was removed by evaporation under nitrogen, and the derivatized sample (butyl esters) was dissolved in 50 µl of methanol/glycerol (1:1, v/v) containing 0.1% octyl sodium sulfate (matrix). A QUATTRO tandem quadrupole mass spectrometer (Fisons Instruments, Inc., Danvers, MA) equipped with a liquid secondary ionization source, and a cesium ion gun was used for the analysis of acylcarnitines. A parent ion scan function at m/z 85 enables the selective detection of acylcarnitine butyl esters. This method of analysis is based on the detection of a common fragment ion of acylcarnitine butyl esters produced by collision-induced dissociation, analogous to the method for methyl esters previously described (Millington et al., 1991). Approximately 2 µl of sample matrix was analyzed, and the data were recorded and processed as described previously (Millington et al., 1991). The final spectra display the relative intensities of ions corresponding to the molecular weights of the individual acylcarnitine butyl esters.

Source of Cells

Normal cell cultures were obtained from the Human Genetic Repository (Camden, NJ). Short-chain acyl-CoA dehydrogenase-deficient cell lines were those described earlier (Amendt et al., 1987; Coates et al., 1988). The diagnoses of patients with medium-chain acyl-CoA dehydrogenase deficiency were established by electron transfer flavoprotein-dependent enzyme assay using octanoyl-CoA as substrate (courtesy of Dr. Paul Coates, Children's Hospital of Philadelphia) and/or by DNA analysis. Two patients were homozygous for the K329E mutation, and three were compound heterozygotes with an I375T mutation on the other allele in one patient and a 4-base deletion in the other two (Ding et al., 1992). The diagnosis of very-long-chain acyl-CoA dehydrogenase deficiency was established by both immunoblot assay and electron transfer flavoprotein-based enzyme assay as described (Yamaguchi et al., 1993). The characterization of electron transfer flavoprotein dehydrogenase-deficient cells has been published (Moon and Rhead, 1987; Rhead et al., 1987). Of the long-chain hydroxyacyl-CoA dehydrogenase-deficient cell lines, one was obtained from the Human Genetic Repository (GM 04081), while others were previously described (Poll-The et al., 1988).


RESULTS

Intermediates Observed during the Oxidation of [^2H(4)]Linoleic Acid

Normal human fibroblasts were incubated with [^2H(4)]linoleic acid in the presence of L-carnitine. Analysis of acylcarnitines by tandem mass spectrometry (tandem MS) yielded patterns representing the molecular species of acylcarnitine butyl esters present in the order of their increasing mass to charge ratio (m/z). Acylcarnitine intermediates observed by tandem MS are products of both fatty acid oxidation and amino acid degradation. The three- and five-carbon species observed in all studies correspond to propionylcarnitine (C(3)) and isovalerylcarnitine and/or 2-methylbutyrylcarnitine (C(5)), which are derived from leucine and isoleucine, respectively. The unlabeled C(4) intermediate corresponds to butyryl- and isobutyrylcarnitines resulting from the oxidation of endogenous unlabeled lipid and valine, respectively. These unlabeled intermediates are always observed when cells are incubated with labeled linoleate. The internal standards [^2H(9)]isovalerylcarnitine ([^2H(9)]C(5)), [^2H(9)]octanoylcarnitine ([^2H(9)]C(8)), and [^2H(3)]palmitoylcarnitine ([^2H(3)]C), added to the incubation mixture, are marked i.s. in the spectra (Fig. 1) and appear at m/z 311, 353, and 459, respectively. Acylcarnitine profiles obtained for normal or the various beta-oxidation-deficient cells (Fig. 1) were consistently similar regardless of the passage number or the time of incubation.

Intermediates detected during the oxidation of [^2H(4)]linoleic acid by normal cells were butyryl ([^2H(4)]C)-, hexanoyl ([^2H(4)]C)-, octanoyl ([^2H(4)]C)-, and decenoylcarnitines ([^2H(4)]C). All of these compounds are formed from intermediates that are substrates of acyl-CoA dehydrogenases (EC 1.3.99.3; butyryl-CoA dehydrogenase, EC 1.3.99.2) (Fig. 1). Intermediates corresponding to substrates of the enoyl-CoA hydratases (EC 4.2.1.17; long-chain-enoyl-CoA hydratase, EC 4.2.1.74), L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and 3-ketoacyl-CoA thiolase (EC 2.3.1.16) were not observed.

The formation of individual metabolites over the course of the incubation was quantitated by tandem mass spectrometry and is depicted in Fig. 2. The relative amounts of labeled metabolites produced throughout the 96-h incubation remained constant as judged by the ratios of 4-cis-decenoylcarnitine to octanoylcarnitine, 2.25, 2.23, and 2.26 at 48, 72, and 96 h, respectively. The ratio of butyrylcarnitine to octanoylcarnitine at the same times was also stable at 0.06, 0.04, and 0.04, respectively.


Figure 2: Time course of acylcarnitine production as observed by incubation of [^2H(4)]linoleate with control and short-chain acyl-CoA dehydrogenase-deficient cells in the presence of L-carnitine. A, concentrations of butyrylcarnitine (bullet), octanoylcarnitine (circle), and decenoylcarnitine () in control cells; B, concentration of butyrylcarnitine in control (bullet) and short-chain acyl-CoA dehydrogenase-deficient (up triangle) cells.



Short-chain acyl-CoA dehydrogenase-deficient cell lines are mainly characterized by a significant elevation of [^2H(4)]butyrylcarnitine levels compared with the internal standard [^2H(9)]C(5) (Fig. 1). As with control cells, there was no apparent accumulation of intermediates containing six or more carbons with or without label. Also, unlabeled C(3)-C(5) acylcarnitines derived from amino acid sources appeared essentially unchanged relative to control cells. The relationship of labeled decenoylcarnitine to octanoylcarnitine was also unchanged, with a ratio of 2.22, as seen in control cells. The actual amounts of [^2H(4)]butyrylcarnitine formed by short-chain acyl-CoA dehydrogenase-deficient cells were 10-11 times greater than the amounts produced by control cells at 48, 72, and 96 h of incubation (Fig. 2B), while concentrations of octanoyl- and decenoylcarnitines were about half those seen in control cells (Table 1).

Medium-chain acyl-CoA dehydrogenase-deficient cells chain-shortened [^2H(4)]linoleic acid to the 10-carbon intermediate as effectively as controls or short-chain acyl-CoA dehydrogenase-deficient cell lines. Labeled intermediates of linoleate beta-oxidation were mainly decenoylcarnitine and octanoylcarnitine, with evidence of further oxidation to hexanoylcarnitine and butyrylcarnitine (Fig. 1). Compared with controls, the concentrations of these four intermediates increased 3.5-, 15-, 7.6-, and 2-fold, respectively (Table 1). As with the other beta-oxidation-deficient cell lines, only intermediates that are substrates of acyl-CoA dehydrogenases were observed. The ratios of labeled decenoylcarnitine to octanoylcarnitine were 0.50, 0.47, and 0.47 at 48, 72, and 96 h of incubation, respectively, compared with the ratio of 2.25 in control cells.

In very-long-chain acyl-CoA dehydrogenase deficiency, the profile of labeled intermediates obtained by tandem MS revealed mainly the presence of the precursor octadecadienoylcarnitine (C) and hexadecadienoylcarnitine (C), in addition to the most prominent metabolite, tetradecadienoylcarnitine (C). These species were not detected in control or short- or medium-chain acyl-CoA dehydrogenase-deficient cell incubations. Labeled intermediates with <10 carbons were barely detectable (Fig. 1).

The labeling pattern observed in long-chain hydroxyacyl-CoA dehydrogenase-deficient cell lines was also unique compared with controls and other defective cell lines (Fig. 1). Metabolites with 8-18 carbons were detected. Hydroxyacylcarnitines corresponding to OH-C and OH-C were observed along with C, C, and C species. There was no significant accumulation of labeled hydroxyacylcarnitines corresponding to 14 carbons or less.

When cell lines deficient in electron transfer flavoprotein dehydrogenase were incubated with labeled linoleic acid, the conversion of substrate to acylcarnitines corresponding to the long-chain C, C, and C species was very low. No formation of intermediates with <14 carbons was detected. This observation suggests a severe impairment of fatty acid oxidation. In addition to the intermediates derived from labeled linoleate, the concentration of unlabeled C(5) acylcarnitines was extremely elevated. The signal for the C(4) unlabeled acylcarnitine was also elevated, but to a lesser extent than that for C(5) (Fig. 1).

[15,16^3H]Palmitate Oxidation

Studies of [15,16-^3H]palmitate oxidation revealed impaired oxidation in most mutant cell lines. [15,16-^3H]Palmitate requires oxidation down to the four-carbon length before ^3H(2)O can be released. Oxidation rates with this compound revealed impaired oxidation relative to controls as follows: short-chain acyl-CoA dehydrogenase-deficient, 39-62%; medium-chain acyl-CoA dehydrogenase-deficient, 25- 40%; very-long-chain acyl-CoA dehydrogenase-deficient, 10-37%; long-chain hydroxyacyl-CoA dehydrogenase-deficient, 7-37%; and electron transfer flavoprotein dehydrogenasedeficient, 7-35%.


DISCUSSION

Intact human fibroblasts were used to study fatty acid oxidation intermediates because relationships among fatty acid oxidation, electron transfer systems, and other interacting pathways of intermediary metabolism are preserved. The integrity of these systems is essential for studying the organization and control of the enzymes involved in mitochondrial beta-oxidation. The availability of beta-oxidation-deficient cell lines from patients with various inherited fatty acid oxidation disorders also provides an opportunity to explore the perturbations of beta-oxidation caused by these enzymatic deficiencies. The resulting alterations in the profile of intermediates might then reveal contributions from enzymes with overlapping chain length specificities, as well as other organizational aspects of the pathway.

Analysis by tandem mass spectrometry of acylcarnitines offers some unique advantages compared with other methods. Since the butyl esters of all acylcarnitines share a common fragment (m/z 85) derived from the carnitine moiety, a parent ion scan of compounds generating that fragment provides only the original mass of each parent acylcarnitine (Millington et al., 1991). This approach obviates the need for purification, isolation, and separation of the metabolites. Furthermore, there exists no doubt about the identity of the intermediates since each has its own unique mass in the entire chain length spectrum.

[^2H(4)]Linoleate (C) was selected as the most useful substrate to probe the pathway because its degradation would require all known enzymes of mitochondrial beta-oxidation including Delta^2,Delta^3-enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (Schulz and Kunau, 1987). Furthermore, each intermediate would contain 4 deuterium atoms on the - and -1-carbons, distinguishing them by tandem MS from intermediates derived from unlabeled lipids and amino acids present in the culture medium.

Apparent Equilibrium of the in Vitro System

The oxidation of C to butyryl-CoA can only occur if all beta-oxidation enzymes are present. The labeling pattern of intermediates in control cells verified the integrity of the pathway (Fig. 1), as did the significant elevation of [^2H(4)]butyrylcarnitine levels in the short-chain acyl-CoA dehydrogenase-deficient cell lines.

The ratios of acyl groups present as acyl-CoA thioesters relative to acylcarnitines reflect their concentrations as well as the kinetic properties of carnitine acyltransferases in intact fibroblasts. Acyl-CoA thioesters, which are produced by mitochondrial beta-oxidation, are formed in and confined to the mitochondria, whereas acylcarnitines can exit from the mitochondria and distribute throughout the incubation medium. Considering the high concentration of L-carnitine used in these incubations and the length of the experiment, the concentrations of acyl-CoAs are most likely negligible compared with the acylcarnitine concentrations, as shown in a similar study using isolated mitochondria from human fibroblasts (Kler et al., 1991).

In control cells, the concentration of intermediates due to the oxidation of linoleate increased continuously over 96 h (Fig. 2). The ratios of decenoylcarnitine to octanoylcarnitine (2.24) and butyrylcarnitine to octanoylcarnitine (0.04) were constant throughout this period of incubation. The ratio of decenoylcarnitine to octanoylcarnitine formed by short-chain acyl-CoA dehydrogenase-deficient cells was also constant at 2.22 throughout the incubation. Even though the amount of C was greatly increased due to short-chain acyl-CoA dehydrogenase deficiency, its concentration relative to the other intermediates also remained constant during the incubation period. Decenoylcarnitine and octanoylcarnitine were both elevated in medium-chain acyl-CoA dehydrogenase-deficient cell lines (3.4- and 15-fold, respectively). However, the ratio of [C] to [C] remained constant at 0.5 during the incubation period. The data suggest that these acylcarnitines are in equilibrium with the accumulated fatty acyl-CoA thioesters present in the mitochondrial matrix.

Overlapping Chain Length Specificities of Acyl-CoA Dehydrogenases

The selection of cell lines deficient in specific enzymes revealed a variety of intermediates that appear to be unique to each deficiency. The labeling of intermediates derived from [^2H(4)]C in beta-oxidation-deficient cell lines provides some insight into the overlapping chain length specificities of acyl-CoA dehydrogenases. Short-chain acyl-CoA dehydrogenase prefers butyryl-CoA as substrate, with lower activity toward hexanoyl-CoA (Dommes and Kunau, 1984; Ikeda et al., 1985). Short-chain acyl-CoA dehydrogenase-deficient cells oxidized [^2H(4)]C to butyryl-CoA, and likely to acetyl-CoA, as reflected by the intermediate patterns and by the constant ratio of butyrylcarnitine to other acylcarnitine intermediates. This conversion is most likely catalyzed by medium-chain acyl-CoA dehydrogenase present in these cells.

There is also a significant overlap between the chain length specificities of medium-, long-, and very-long-chain acyl-CoA dehydrogenases as evidenced by the oxidation of [^2H(4)]C in medium-chain acyl-CoA dehydrogenase deficiency. The elevations of acylcarnitine levels corresponding to C and C, in addition to C, possibly reflect the dehydrogenation by long- and very-long-chain acyl-CoA dehydrogenases. Incubation of medium-chain acyl-CoA dehydrogenase-deficient cells with labeled linoleic acid revealed good conversion to octanoyl-, hexanoyl-, and butyrylcarnitines, suggesting that medium-chain acyl-CoA dehydrogenase-deficient cells are capable of converting 4-cis-decenoyl-CoA, which is a preferred substrate for medium-chain acyl-CoA dehydrogenase (Dommes and Kunau, 1984), into shorter chain length intermediates. These conversions are likely due to the activity of long-chain acyl-CoA dehydrogenase with medium-chain substrates.

The formation of C in very-long-chain acyl-CoA dehydrogenase deficiency is also of interest. This labeled intermediate dominates the acylcarnitine profile of very-long-chain acyl-CoA dehydrogenase-deficient cells incubated with [^2H(4)]linoleate and is accompanied by lesser amounts of C and C carnitines. This observation suggests that the C intermediate is a preferred substrate for very-long-chain acyl-CoA dehydrogenase. Further investigations with proven long-chain acyl-CoA dehydrogenase-deficient cell lines should clarify the overlapping chain length specificities of these two enzymes toward unsaturated substrates. However, since most long-chain acyl-CoA dehydrogenase-deficient cell lines described to date have been shown to be very-long-chain acyl-CoA dehydrogenase-deficient instead (Yamaguchi et al., 1993), these studies must await the identification of long-chain acyl-CoA dehydrogenase-deficient patients.

In addition to L-3-hydroxyacylcarnitines, which accumulated during the incubation of cells deficient in L-3-hydroxyacyl-CoA dehydrogenase, the most prominent intermediates were the precursor C, C, and C. These latter species were also detected in incubations with both very-long-chain acyl-CoA dehydrogenase-deficient and electron transfer flavoprotein dehydrogenase-deficient cells. A possible explanation for this profile is that long-chain L-3-hydroxyacyl-CoA thioesters strongly inhibit acyl-CoA hydratases (He et al., 1992), which consequently inhibit acyl-CoA dehydrogenases, thereby causing an accumulation of acyl-CoA thioesters (Thorpe, 1990).

Intermediate Channeling and Organization of the Mitochondrial beta-Oxidation Enzymes

The enzymes of mitochondrial beta-oxidation appear to be organized into two distinct systems (Fig. 3). The first system is the long chain-specific system, which includes very-long-chain acyl-CoA dehydrogenase and the trifunctional enzyme that harbors the long-chain activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase. This system also requires carnitine palmitoyltransferase I, carnitine/acylcarnitine translocase, and carnitine palmitoyltransferase II. All of these enzymes are known to be bound to the inner mitochondrial membrane (Uchida et al., 1992; Izai et al., 1992), with the exception of carnitine palmitoyltransferase I, which is bound to the outer membrane (Murthy and Pande, 1987). The other beta-oxidation system, which has preference for medium- and short-chain substrates, consists of soluble enzymes and is located in the mitochondrial matrix (Schulz, 1991).


Figure 3: Model of the functional and physical organization of beta-oxidation enzymes in mitochondria. A, beta-oxidation system active with long-chain acyl-CoAs; B, beta-oxidation system active with medium- and short-chain acyl-CoAs. T, carnitine/acylcarnitine translocase; CPT II, carnitine palmitoyltransferase II; AD, acyl-CoA dehydrogenase; EH, enoyl-CoA hydratase; HAD, L-3-hydroxyacyl-CoA dehydrogenase; KT, 3-ketoacyl-CoA thiolase; VLC, very-long-chain; LC, long-chain; MC, medium-chain; SC, short-chain.



It appears that long-chain fatty acids are efficiently chain-shortened to the medium-chain level in both normal cells and cells defective in short- or medium-chain activities as evidenced by the absence of significant amounts of the longer chain length acylcarnitine species ( Fig. 1and Table 1).

Intermediates detected during the incubation of normal cells or cells defective in one of the mitochondrial matrix enzymes such as short- or medium-chain acyl-CoA dehydrogenase (Fig. 1) were of medium- or short-chain length and represent substrates for those acyl-CoA dehydrogenases. Long-chain intermediates were detectable only in very-long-chain acyl-CoA dehydrogenase-, long-chain hydroxyacyl-CoA dehydrogenase-, and electron transfer flavoprotein dehydrogenase-deficient cell lines (Fig. 1), which are all membrane-bound proteins and belong to the long chain-specific system. The failure to detect long-chain intermediates, including intermediates that are substrates of acyl-CoA dehydrogenase, in the intact cell system suggests that enzymes involved in the membrane system exist in an organized arrangement. As a consequence of this organization, substrates appear to be channeled without the accumulation of free intermediates. In such a system, no particular reaction would be rate-limiting. Inhibition of any of the enzymes involved in this organization (such as very-long-chain acyl-CoA dehydrogenase, long-chain hydroxyacyl-CoA dehydrogenase, or electron transfer flavoprotein dehydrogenase) appears to inhibit the complete pathway (Fig. 1). Fong and Schulz(1978) showed that the inhibition of thiolases paralleled the inhibition of palmitoylcarnitine-supported respiration. Also, respiration supported by either palmitoyl-CoA or octanoate was inhibited by 4-pentenoate in a nearly identical fashion, suggesting that the thiolase-catalyzed step is rate-limiting. Incremental inhibition of carnitine palmitoyltransferase I by 2-tetradecylglycidoyl-CoA resulted in almost parallel decreases in the respiration rates with either palmitoyl-CoA or docosahexaenoyl-CoA as substrate, (^2)suggesting a tight coupling between carnitine palmitoyltransferase I and beta-oxidation. Kerner and Bieber(1990) showed that an affinity-purified preparation of mitochondrial carnitine palmitoyltransferase contained activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. All of these observations agree with the notion that beta-oxidation enzymes exist as organized complexes that facilitate intermediate channeling.

In the matrix, the observed medium- and short-chain intermediates, whether formed in normal cells or in lines defective in acyl-CoA dehydrogenases, were all substrates of acyl-CoA dehydrogenases. Intermediates corresponding to substrates for enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, thiolase, enoyl-CoA isomerase, and 2,4-dienoyl-CoA reductase were not observed. The lack of product-precursor relationships between intermediates produced by matrix enzymes acting in sequence as shown in this study and others (Stanley and Tubbs, 1975; Kler et al., 1991) agrees with the idea that the matrix enzymes exist in an organized or at least in a nonrandom arrangement that facilitates the cooperation between these enzymes. However, the observed accumulation of medium- and short-chain intermediates, which are substrates of acyl-CoA dehydrogenases, suggests that the transfer of intermediates from thiolase to acyl-CoA dehydrogenases in the matrix does not proceed by complete channeling. The reason for this partial channeling could be related to the involvement of only one 3-ketoacyl-CoA thiolase with the three acyl-CoA dehydrogenases that may be unable to associate into a single complex.

In this study, we have utilized intact normal human fibroblasts and fibroblasts defective in various beta-oxidation enzymes to investigate the organization and overlapping chain length specificities of the enzymes involved in this metabolic pathway. Two distinct situations of intermediate transfer can be discerned. A complete substrate channeling seems to take place on the long chain-specific enzymes associated with the mitochondrial membrane. Partial channeling seems to occur on the matrix enzymes that are responsible for the oxidation of medium- and short-chain fatty acyl-CoA thioesters. The accumulation of only intermediates that are substrates of acyl-CoA dehydrogenases appears to be due to the incomplete channeling between the single 3-ketoacyl-CoA thiolase and the multiple acyl-CoA dehydrogenases present in the matrix. 3-Ketoacyl-CoA thiolase and the several acyl-CoA dehydrogenases might be unable to form a single complex.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HD24908 (to C. R. R.) and DK33289 (to W. J. R.); State of North Carolina, Division of Maternal and Child Health, Department of Environmental Health and Natural Resources Grant C05070; General Clinical Research Center Grant M-01-RR-30; and memorial funds for H. L. Holtkamp and Kristen Gould (to Duke Medical Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Duke University Medical Center, Pediatrics Biochemical Genetics, P. O. Box 14991, Research Triangle Park, NC 27709. Tel.: 919-549-0445; Fax: 919-549-0709.

(^1)
The abbreviations used are: BSA, bovine serum albumin; tandem MS, tandem mass spectrometry.

(^2)
M. A. Nada and H. Schulz, unpublished data.


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