(Received for publication, July 21, 1994; and in revised form, October 17, 1994)
From the
The accumulation of -oxidation intermediates was studied by
incubating normal and
-oxidation enzyme-deficient human
fibroblasts with [
H
]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
-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
-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.
Investigations of the control and organization of the enzymes
involved in mitochondrial fatty acid -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
-oxidation
intermediates is not easily resolved. Similarly, the rate of oxidation
of radiolabeled fatty acids, such as
[1-
C]palmitate,
[9,10-
H
]palmitate, etc., can, at
best, reveal a reduced rate of
-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-H
]linoleate
([
H
]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
-oxidation.
Skin fibroblasts (0.12-0.16 mg of protein) were
subcultured in 75-cm 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
, 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
[H
]isovalerylcarnitine
([
H
]C
), 20 pmol of
[
H
]octanoylcarnitine
([
H
]C
), and 40 pmol of
[
H
]palmitoylcarnitine
([
H
]C
) as internal
standards. The concentrations of acylcarnitines corresponding to chain
lengths of C
, C
, and C
were
determined based on their intensities relative to the internal standard
[
H
]C
. The concentrations
of C
and C
were measured relative to the
concentration of the internal standard
[
H
]C
. Those of
C
-C
were determined based on their
relative concentrations to
[
H
]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
[H
]linoleic acid. The signals
represent the molecular ions of acylcarnitine butyl esters detected by
tandem MS. [
H
]Isovalerylcarnitine,
[
H
]octanoylcarnitine, and
[
H
]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 [H
]palmitate
were determined as described (Moon and Rhead, 1987), except that the
concentration of [15,16-
H]palmitate was 80
µM. Fibroblast cultures from five normal controls and each
patient were assayed in duplicate three to five times.
Intermediates detected during the oxidation of
[H
]linoleic acid by normal cells were
butyryl ([
H
]C
)-,
hexanoyl ([
H
]C
)-,
octanoyl ([
H
]C
)-, and
decenoylcarnitines
([
H
]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
[H
]linoleate with control and
short-chain acyl-CoA dehydrogenase-deficient cells in the presence of L-carnitine. A, concentrations of butyrylcarnitine
(
), octanoylcarnitine (
), and decenoylcarnitine (
) in
control cells; B, concentration of butyrylcarnitine in control
(
) and short-chain acyl-CoA dehydrogenase-deficient (
)
cells.
Short-chain acyl-CoA
dehydrogenase-deficient cell lines are mainly characterized by a
significant elevation of
[H
]butyrylcarnitine levels compared
with the internal standard [
H
]C
(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
-C
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
[
H
]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 [H
]linoleic
acid to the 10-carbon intermediate as effectively as controls or
short-chain acyl-CoA dehydrogenase-deficient cell lines. Labeled
intermediates of linoleate
-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
-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
acylcarnitines was extremely elevated. The signal for the C
unlabeled acylcarnitine was also elevated, but to a lesser extent
than that for C
(Fig. 1).
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 -oxidation. The availability of
-oxidation-deficient cell lines from patients with various
inherited fatty acid oxidation disorders also provides an opportunity
to explore the perturbations of
-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.
[H
]Linoleate (C
)
was selected as the most useful substrate to probe the pathway because
its degradation would require all known enzymes of mitochondrial
-oxidation including
,
-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.
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
-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.
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
[H
]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
[
H
]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).
Figure 3:
Model
of the functional and physical organization of -oxidation enzymes
in mitochondria. A,
-oxidation system active with
long-chain acyl-CoAs; B,
-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, ()suggesting a tight
coupling between carnitine palmitoyltransferase I and
-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
-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 -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.