Since the first demonstration of the presence of a fatty acid
-oxidation system in rat liver peroxisomes by Lazarow and de Duve (1) , it has become increasingly apparent that peroxisomes play
an important role in a number of lipid metabolic pathways. The
peroxisomal disorders, a group of inherited metabolic
disorders(2) , derive from defects of peroxisomal biogenesis
and/or dysfunction of peroxisomal enzymes. Numerous biochemical
abnormalities may result: decreased red blood cell
plasmalogens(3) , increased plasma phytanic acid(4) ,
pristanic acid(5) , pipecolic acid(6) , and very long
chain fatty acids (7) , and increased urinary medium and long
chain dicarboxylic acids (8) , cholestanoic acids(9) ,
eicosanoids(10) , epoxy-dicarboxylic acids (11) , and
2-hydroxy acids(12) . The abnormal metabolism of these
compounds provides evidence for the importance of peroxisomes in many
catabolic and biosynthetic lipid pathways. In addition, analyses of
these compounds may serve as diagnostic tools for the detection of
peroxisomal disorders. Fast atom bombardment mass spectrometric
(FAB-MS) (
)analysis of cholestanoic acids in urine is useful
in the diagnosis of many peroxisomal
-oxidation
defects(13, 14, 15) . In evaluating more than
100 urine samples from patients with peroxisomal disorders, we noted
several ions in the FAB-MS spectra that appear to be associated with
generalized peroxisomal disorders and have not been previously
reported. The ions were rarely observed in spectra obtained from normal
or cholestatic liver disease control samples. Our experience suggested
that ions at m/z 489 and 505 represent novel compounds
accumulating in peroxisomal disease, characterization of which may be
useful both in understanding of the pathogenesis of these diseases and
as a diagnostic tool. In this study we are reporting the structures of
the compounds represented by these ions.
EXPERIMENTAL PROCEDURES
Materials
All solvents were obtained from Curtin Matheson, Wilmington,
MA, and were Omnisolv or HPLC grade. All glassware was silanized with
5% dimethylchlorosilane (Sigma) and washed thoroughly with toluene and
methanol before use. Extract Clean/RC C18 cartridges (0.1 and 0.5 g)
(Alltech Associates, Deerfield, IL) were washed and used as described
previously(15) . Octadecylsilane chromatography material,
Sepralyte (Analytichem International, Harbor City, CA), was similarly
prepared. The lipophilic anion exchange gel diethylaminohydroxypropyl
(DEAP)-Sephadex LH-20 was synthesized from Sephadex LH-20 using the
method described by Alme et al.(16) . For this
synthesis epichlorohydrin and diethylamine were obtained from Sigma.
Titration of the prepared gel with 0.1 M HCl in methanol
demonstrated it had an exchange capacity of 1.42 mEq/g. The gel was
converted to the acetate form by washing with 1 M acetic acid
in 72% ethanol followed by 72% ethanol to neutrality. Volume
requirements for elution of compounds of interest were determined by
the use of appropriate bile acid standards. Type HP-2
-glucuronidase (crude solution from Helix Pomatia), anhydrous
tetrahydrofuran, acetyl chloride, Sigma Sil A, acetic anhydride ACS,
hexamethyldisilazane (HMDS); trimethylchlorosilane (TMCS); N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA);
pyridine ACS, bile acid standards, and erthro- and threo-9,10-dihydroxy
stearic acid were obtained from Sigma. Econo columns for liquid
chromatography were obtained from Bio-Rad. The DB-17 fused silica GC
capillary column (30 m
0.25 mm inside diameter
0.25
µm phase thickness) was from J & W Scientific, Chicago, IL.
Methods
Continuous Flow (CF)/FAB-MS
Urine samples (2 ml) from normal children, children with
cholestatic liver disease, and patients with established and suspected
peroxisomal disease were extracted and analyzed by
CF/FAB-MS(15) . Direct injection-CF/FAB-MS and micro high
performance liquid chromatography CF/FAB-MS were carried out as
described previously (15) using a VG70-250 S.E. double
focusing mass spectrometry system linked to a Brownlee Labs
Microgradient solvent delivery system and incorporating a 1-mm inside
diameter
100-mm column packed with 3 µm ODS Hypersil
(Keystone Scientific, Bellafont, PA) for HPLC.
Sodium Borodeuteride Reduction of Ketones
Urine extracts were reduced by the method of Bjorkhem et
al.(17) .
Extraction Procedures
A schematic of the isolation procedure is shown in Fig. 1. Urine was collected from patients with m/z 489
as an intense ion in the FAB-MS spectra, and an aliquot of up to 500 ml
was extracted. Columns (0.7-1.5 cm inside diameter, dependent on
sample size) were packed with octadecylsilane bonded phase (1 g/20 ml
of urine) and washed with methanol (10 ml/g) and distilled water (10
ml/g) prior to application of sample. Nitrogen gas pressure was applied
to obtain a flow rate which did not exceed 10 ml/min. The column was
washed several times with water (60 ml/g of column packing) and eluted
with methanol (10 ml/g of column packing).
Figure 1:
Schematic representation of the
procedures used in the isolation and analysis of the hydroxylated fatty
acids.
Chromatographic Conditions
DEAP-Sephadex LH-20 (acetate form) was packed (0.7
10
cm) in Bio-Rad Econo columns under nitrogen gas pressure (0.3-0.5
kg/cm
) and the column equilibrated in 70% methanol. The
methanol eluate from the C18 extraction step was diluted with water to
70% methanol and passed through the column. Buffer systems described by
Alme et al.(16) were used to elute the compounds of
interest and separate them from the bulk of other compounds in the
extract. Three fractions were collected, corresponding to those
described by Alme et al.(16) for the (a)
sterols, (b) unconjugated and glycine/taurine-conjugated bile
acids, and (c) sulfate/glucuronide-conjugated bile acids. The
columns were successively washed with 10 ml of 70% ethanol (fraction
a), followed by 10 ml of 0.15 M acetic acid in 70% ethanol (pH
adjusted to 6.6 with ammonium hydroxide) (fraction b). The compounds of
interest were then eluted along with other sulfated and glucuronic acid
conjugates with 10 ml of 0.3 M acetic acid in 70% ethanol (pH
adjusted to 9.6 with ammonium hydroxide) (fraction c). This fraction
was evaporated to a small volume, diluted with water to less than 5%
methanol, and desalted on a C18 column (1 g/50 ml of urine extract).
Solvolysis
Solvolysis was carried out in 20 ml of freshly prepared
tetrahydrofuran-4 M H
SO
(100:0.15,
v/v) at 50°C for 3.5 h. Products were recovered by diluting the
solvolysis solution to less than 5% tetrahydrofuran with water and
desalted on a C18 column. The methanol eluate was evaporated,
redissolved in a small volume of 70% methanol, and chromatographed
again on a DEAP-Sephadex LH-20 column. Compounds conjugated to
glucuronic acid were now concentrated in this final fraction.
Glucuronide Fraction: Purification and Derivatization for
Gas Chromatography-Mass Spectrometry
The glucuronide fraction was evaporated and subjected to
enzyme hydrolysis with
-glucuronidase or further purified as
follows. The sample was redissolved in a small volume of water and
applied to a 0.5 g of Extract Clean/RC C18 cartridge. The cartridge was
washed with 30 ml of water and eluted with 4 ml of each of 10, 20, 40,
50, 60, 70, 80, and 100% methanol in water. The elution profile was
monitored using direct injection CF/FAB-MS. The compounds of interest
eluted in the 50 and 60% methanol fractions. These fractions were then
evaporated and derivatized for GC-MS analysis.In order to provide
comprehensive characterization of the glucuronide fatty acids four
derivatization procedures were used as shown in Fig. 2. Samples
were derivatized as follows: scheme 1, permethylated (PM)(18) ,
providing molecular weight data and information on the number of
derivatization sites; scheme 2, permethylated (18) followed by
methanolysis and trimethylsilylation (PM-methanolysis-TMS), providing
information about the site of glucuronide conjugation; scheme 3,
subjected to methanolysis before trimethylsilylation
(methanolysis-TMS), providing high mass fragment ions useful in the
characterization of the aglycone structure; and scheme 4, subjected to
treatment with
-glucuronidase prior to methylation of the carboxyl
group and trimethylsilylation of the hydroxyl groups, providing
confirmatory evidence of the glucuronic acid conjugation.
Figure 2:
Schematic representation of the
derivatization methods used in the analysis of the hydroxylated fatty
acids by GC-MS.
Methanolysis
Methanolysis was carried out using an
adaptation of the method of Wiesner and Sweeley(19) . The
methanolysis reagent was freshly prepared by slowly adding acetyl
chloride (50 µl) to methanol (1 ml)(20) . The samples were
transferred to silanized microcapillary tubes (approximately 75 mm
1.6 mm) with one end sealed. The tubes were placed in plastic
cups and the solvent evaporated in a Speed-Vac under vacuum. After
methanolysis reagent (30 µl) was added, the tubes were sealed in a
flame and placed in an oven at 90°C overnight. After cooling the
capillaries were scored and broken, and the reagent was evaporated
under vacuum.
TMS
The methylated samples were trimethylsilylated
in sealed microcapillary tubes with 10 µl of Sigma Sil-A at
60°C for 30 min. Deuterated derivatives were formed using 10 µl
of a mixture of d
-HMDS, d
-TMCS, and d
-BSTFA in dry
pyridine (5:1:1:1, v/v/v/v) at 60°C for 30 min.
Glucuronidase Treatment
The fractions were
dissolved in a minimum of 5 ml 0.2 M acetate buffer, pH 4.5,
and 100 µl of
-glucuronidase was added (approximately 1000
IU). After shaking overnight at 37°C, an additional aliquot of
-glucuronidase was added and the incubation repeated. In order to
extract the aglycone, the hydrolysis solution was diluted with water
and twice applied to a prepared C18 column (2 g). The column was washed
with water (120 ml) and eluted with methanol (30 ml).
Methylation
The aglycone was methylated in 30
µl of methanolysis reagent, prepared as described above, heated at
60°C for 30 min.
GC-MS
A Finnigan 4500 quadrupole GC-MS system equipped with a
Teknivent Vector/Two data system and Hewlett Packard 5890 GC was used
for these studies. A fused silica DB-17 capillary column was used. The
oven temperature was held at 180°C for 2 min and then programmed at
10°C/min to 330°C, where it was held for 2 min. Chemical
ionization (CI) spectra were obtained using ammonia gas (0.7 Torr) with
source temperature at 180 °C. Electron impact (EI) spectra were
obtained at 70 eV with the source temperature at 190 °C.
RESULTS
FAB-MS
FAB-MS spectra from pooled normal control urine and urine
from a patient with generalized peroxisomal disease are shown in Fig. 3. The m/z 489 ion was one of the 10 most intense
ions in the FAB-MS spectra of urine from 45 of 60 well characterized
generalized peroxisomal disorder patients studied. It was also observed
as one of the 10 most intense ions in the FAB-MS spectra of 1 of 12
patients with bifunctional protein enzyme deficiency, (
)a
single enzyme defect in the peroxisomal
-oxidation pathway.
Preliminary results indicated that the ion at m/z 489 was not
observed as one of the 10 most intense ions in FAB-MS spectra of 15
normal children or in 15 children with cholestatic liver disease or
hepatic failure. Less intense ions which also appeared in some
peroxisomal patients' urine samples included m/z 491,
487, 463, 461, 459, 507, 505, and 596. Compounds which correspond to
these ions are described in this report.
Figure 3:
Negative ion CF/FAB mass spectra of a
pooled control urine and a urine sample from a patient with generalized
peroxisomal disorder.
The major FAB-MS ions of
interest at m/z 489 and 505, which represent (M -
H)
ions for compounds of M
490
and 506, respectively, do not shift after reduction with sodium
borodeuteride, suggesting these compounds do not contain a reducible
ketone group. They consistently elute from DEAP-Sephadex LH-20 in the
fraction containing sulfate and glucuronic acid conjugates. Monitoring
of anion exchange chromatography by CF/FAB-MS showed that sulfated bile
acids are successfully solvolyzed using the solvolysis conditions
described. In contrast, the ions of interest at m/z 489 and
505 are unaffected by solvolysis but disappear with glucuronidase
treatment.
Characterization of Hydroxylated Fatty Acids
Two purified fractions, the 50 and 60% methanol elution bands
from C18 chromatography, were found to contain the bulk of the
compounds of interest. Fig. 4shows TIC chromatograms from
CI/GC-MS of these fractions after PM-methanolysis-TMS treatment. The
compounds identified are listed in Table 1. Characterization of
the major species is discussed below. Mass spectral data used in the
characterization of the major compound, 12,13-dihydroxy-9-octadecenoic
acid, are shown in Fig. 5. Similar data were obtained and
utilized in the characterization of the other compounds listed in Table 1, although only the EI spectra of the methanolysis-TMS
derivatives are shown ( Fig. 6and Fig. 7). Specification
of the site of the double bonds is tentative, although there are strong
indications in the mass spectra obtained for the assignments given.
Figure 4:
TIC plot of a CI/GC-MS analysis of
dihydroxy and trihydroxy fatty acids. 50 and 60% methanol fractions
from octadecylsilane chromatography are indicated. The peaks are
identified in Table 1.
Figure 5:
EI and ammonia CI mass spectra of the
glucuronide conjugated 12,13-dihydroxy-9-octadecenoic acid A,
as a permethylated derivative; B, after methanolysis and
trimethylsilylation; and C, after permethylation,
methanolysis, and
trimethylsilylation.
Figure 6:
EI mass spectra of methyl trimethylsilyl
derivatives of A, 9,10-dihydroxyoctadecanoic acid; B,
9,10-dihydroxy-12-octadecenoic acid (major isomer); C,
9,10-dihydroxy-12-octadecenoic acid (minor isomer); D,
12,13-dihydroxy-6,9-octadecadienoic acid; and E,
15,16-dihydroxy-9,12-octadecadienoic acid.
Figure 7:
EI mass spectra of methyl trimethylsilyl
derivatives of A, 7,8-dihydroxyhexadecanoic acid; B,
7,8-dihydroxy-10-hexadecenoic acid (major isomer); C,
7,8-dihydroxy-10-hexadecenoic (minor isomer); D,
10,11-dihydroxy-7-hexadecenoic acid; and E,
7,8-dihydroxy-4,10-hexadecadienoic acid.
Permethylation
Ammonia CI mass spectra of permethylated dihydroxy C18 fatty
acid glucuronides give prominent (M + NH
)
ions at m/z 594, 592 (Fig. 5A), and 590
for the saturated, monounsaturated, and diunsaturated forms,
respectively. Similar ions are seen at m/z 464, 462, and 460
in spectra from dihydroxy C16 fatty acids. The EI spectra are dominated
by ions arising from the glucuronide moiety, although ions
corresponding to aglycone fragments are also observed.
Deuterium-labeled TMS Derivatives
Derivatization with d
-TMS reagents
produces incremental mass changes dependent on the number of TMS groups
in a fragment ion and are useful in assigning fragment ion structures.
As an example the fragment ion seen at m/z 155 in
methanolysis-TMS EI spectra of 9-hydroxylated fatty acids (Fig. 6B) did not change with deuterium labeling of the
TMS derivative. This is consistent with the proposed ion structure of
(O-CH-(CH
)
-C=O)
.
In contrast, after d
-TMS derivatization, the ions m/z 173, 299, and 270 in the EI spectra shown in Fig. 5B all shifted 9 mass units, consistent with a
single TMS group in the ions, and m/z 275 shifted 18 mass
units, consistent with the presence of two TMS groups. Similar
assessments were made in our analysis of the data for all the hydroxy
fatty acids and in each case the incremental change after d
-TMS derivatization is consistent with the
proposed structure.
Glucuronidase Treatment
Spectra obtained following glucuronidase treatment are
similar to those found with methanolysis-TMS analysis, although
recoveries were poor and only the major compounds are observed.
GC-MS Analysis: Characterization of Major Species
12,13-Dihydroxy-9-octadecenoic Acid (2 Isomers)
PM
The CI spectra of the permethylated
derivative of the major isomer as its intact glucuronide has as base
ion m/z 592 ((M + NH
)
) (Fig. 5A). The ion at m/z 325 corresponds to a
loss of glucuronic acid. The EI spectrum is dominated by ions arising
from the glucuronide moiety but also shows ions due to the aglycone
fragment and fragmentation between the glucuronic acid and methyl
substituted hydroxyl groups at m/z 377.
Methanolysis-TMS
The CI spectra obtained following
methanolysis and trimethylsilylation show an intense ion at m/z 383 (MH
- 90), with ions at m/z 473 (MH
), 293 (MH
- (2
90)), and 490 ((M + NH
)
),
confirming the number of OTMS groups in the compound (Fig. 5B). In the EI spectra, abundant fragment ions at m/z 173 and 299 arise from fragmentation between the vicinal
TMS hydroxyl groups. The position of the double bond at C-9 would
direct fragmentation adjacent to the oxygenated carbon atom closest to
the double bond(22) . This is consistent with the formation of
the ion at m/z 275. The ion at m/z 270 is due to the
olefinic fragment from this cleavage with transfer of a TMS group to
the carboxylate radical site.
PM-Methanolysis-TMS
Evidence for glucuronide
linkage at the C-12 and C-13 positions is apparent in the
PM-methanolysis-TMS products, although the major species appears to be
the C-13-glucuronide. The CI and EI spectra from the 13-glucuronide
product (i.e. the 12-O-methyl, 13-OTMS-9-octadecenoic
methyl ester) and proposed fragmentation are shown in Fig. 5C.
GC-MS Analysis: Characterization of Minor C18
Dihydroxy Fatty Acids
9,10-Dihydroxyoctadecanoic Acid
Methanolysis-TMS
The CI spectrum of this
compound contains major ions at m/z 475 (MH
)
and 385 (MH
- 90). In the EI spectrum (Fig. 6A) the abundant ions at m/z 259 and 215
result from cleavage between the two OTMS substituted carbons with
little other fragmentation. The spectra and retention times obtained
are identical to those obtained from the methyl ester-TMS ether
derivative of standard threo-9,10-dihydroxyoctadecanoate and well
separated from the standard erythro isomer.
PM-Methanolysis-TMS
The EI spectra indicate that
the glucuronide is principally found at C-10. With a TMS group
substituted for the original glucuronide, the most intense ion in the
spectra is at m/z 215 which is the
-end fragment ion
following cleavage between the vicinal methyl and TMS ethers.
9,10-Dihydroxy-12-octadecenoic Acid (2 Isomers)
Methanolysis-TMS
Derivatives of the two isomers
give very similar mass spectra. In the CI spectra ions are observed at m/z 473 (MH
) and m/z 490 ((M +
NH
)
). An intense ion at m/z 383
and a smaller one at m/z 293 correspond to the loss of one and
two trimethylsilanols, respectively. The EI spectra of both isomers are
also similar (Fig. 6, B and C) with prominent
ions at m/z 259 and 213 due to cleavage between the vicinal
trimethylsilyl ethers. A weak ion at m/z 332 arises by
migration of a OTMS group to the carboxylate radical site.
Fragmentation, to give ions at m/z 361 and 271 (Fig. 6B), is probably directed by the presence of the
double bond located one methylene unit away from the oxygenated carbon
atom(22) , as are the ions at m/z 103 and 129 which
correspond to
(CH
=O=Si(CH
)
)
and
(CH
=CH-CH=O-Si(CH
)
)
.
The lack of these ions in the second isomer may indicate the double
bond is in a different position.
PM-Methanolysis-TMS
Evidence for substitution with
the glucuronic acid on both the 9 and 10 hydroxyl groups is observed in
the mass spectra of PM-methanolysis-TMS derivatives. The first is
characterized by an intense ion at m/z 259, consistent with
the glucuronide at C-9, and the second by an ion at m/z 213,
consistent with the glucuronide at C-10 (not shown).
12,13-Dihydroxy-6,9-octadecadienoic,
15,16-Dihydroxy-9,12-octadecadienoic, and
9,10-Dihydroxy-12,15-octadecadienoic Acids
Methanolysis-TMS
The EI spectra of the
diunsaturated dihydroxy C18 fatty acids are shown in Fig. 6, D and E. Fragmentation is consistent with the
structures of 12,13-dihydroxy-6,9-octadecadienoic acid and
15,16-dihydroxy-9,12-octadecadienoic acid. In each case fragmentation
occurred between the vicinal TMS ethers with the saturated fragment
giving the most intense signal. Saturated fragments that include both
vicinal TMS ethers also occur, presumably because fragmentation is
directed by the presence of a double bond located one methylene unit
away. Good spectra of the 9,10-dihydroxy-12,15-octadecadienoic acid
isomer could not be obtained due to interfering ions from other
chromatographic peaks. Identification of this compound was principally
made on the basis of the PM-methanolysis-TMS data (not shown).
PM-Methanolysis-TMS
Data from these derivatives
support the observation that at least three species of diunsaturated
dihydroxy fatty acids are present. In CI spectra, base ions are seen at m/z 430 ((M + NH
)
), along
with ions at m/z 413 (MH
), 323 (MH
- HOTMS), 381 (MH
-
OCH
), and 291 (MH
-
OCH
- HOTMS). The EI spectra in each case show
an intense ion which is attributable to a saturated fragment ion
containing the TMS ether and suggest that the glucuronide is located on
C-15 of 15,16-dihydroxy-9,12-octadecadienoic acid and on C-9 of the
9,10-dihydroxy-12,15-octadecadienoic acid. The data obtained do not
permit us to ascertain the position of the glucuronide on
12,13-dihydroxy-6,9-octadecadienoic acid.
GC-MS Analysis: Characterization of Dihydroxy
C16 Fatty Acids
7,8-Dihydroxyhexadecanoic Acid
Methanolysis-TMS
The CI spectrum shows prominent
ions at m/z 447 (MH
) and 357 (MH
- HOTMS). The EI spectrum contains ions arising from
cleavage between the vicinal TMS ethers, m/z 215 and 231, with
both saturated ions having similar intensity (Fig. 7A).
PM-Methanolysis-TMS
The EI spectra of this
derivative suggests that either of the hydroxyl groups can be
glucuronidated giving rise to two spectra containing intense ions at m/z 231 or 215. These correspond to a glucuronide at C-7 and
C-8, respectively.
7,8-Dihydroxy-10-hexadecenoic Acid (2 Isomers)
Methanolysis-TMS
The CI spectra of these
derivatized compounds show abundant ions at m/z 445
(MH
) and 355 (MH
- HOTMS). The
EI spectra of the major isomer shows fragment ions arising from
cleavage between the vicinal TMS ethers, with the saturated fragment
ion at m/z 231 providing a more intense ion than the olefinic
fragment ion at m/z 213 (Fig. 7B). The weak
ion at m/z 333 is presumably due to cleavage between C-8 and
C-9. Loss of a HOTMS group gives rise to the ion at m/z 243.
The abundance of these two ions, in comparison to similar ions seen in
the saturated 7,8-dihydroxyhexadecanoic acid, strongly suggests the
double bond is located at C-10, one methylene unit from the
hydroxylated carbon. A minor isomer of this compound was also observed
and gave a similar spectrum (Fig. 7C), although high
mass fragment ions were less intense relative to the ion at m/z 73.
PM-Methanolysis-TMS
The EI spectrum shows a base
peak at m/z 231 and ions at m/z 275 and 185 arising
from double bond-directed cleavage between C-8 and C-9. This is
consistent with the glucuronide at C-7.
10,11-Dihydroxy-7-hexadecenoic Acid
Methanolysis-TMS
The CI spectrum is similar to
those seen for 7,8-dihydroxy-10-hexadecenoic acid isomers, with the
inclusion of a weak (M + NH
)
ion at m/z 462. The EI spectrum (Fig. 7D) shows ions
arising from cleavage between vicinal TMS ethers with the saturated
fragment ion at m/z 173 more abundant than the olefinic
fragment ion at m/z 271. Abundant ions at m/z 275 and
185 arise from cleavage between C-9 and C-10, probably directed by a
double bond at C-7. An intense rearrangement ion, m/z 242,
also arises from cleavage at this position with migration of a TMS
group to the carboxyl carbonyl.
PM-Methanolysis-TMS
Two compounds are seen with
spectra consistent with a glucuronide at C-10 and C-11, respectively.
In the former, ions at m/z 217 and 271, arise from cleavage
between C-9 and C-10 (
-fragment) and C-10 and C-11 (olefinic
fragment), respectively. In the latter the
-fragment ion, m/z 217, is also apparent but base peak is m/z 173 due to the
glucuronide at C-11.
7,8-Dihydroxy-4,10-hexadecadienoic Acid
Methanolysis-TMS
Only one diunsaturated
dihydroxy C16 fatty acid is observed. The CI spectrum shows prominent
ions at m/z 443 (MH
), 353 (MH
- HOTMS) and 263 (MH
- (2
HOTMS)), with a weak ion at m/z 460 ((M +
NH
)
). The EI spectrum obtained from the
methanolysis-TMS derivative is shown in Fig. 7E. This
spectrum contains fragment ions arising from cleavage between the
vicinal OTMS groups, at m/z 229 and 213, and on either side of
them, at m/z 315 and 331. Loss of a HOTMS group from each of
the latter fragment ions, gives rise to ions at m/z 225 and
241, respectively. The observed cleavage on both sides of the vicinal
OTMS groups suggests the double bonds may occur at C-4 and C-10, one
methylene unit away from C-7 and C-8, respectively.
PM-Methanolysis-TMS
An abundant ion at m/z 229, consistent with fragmentation of the substituted vicinal diol
with the OTMS group at C-7, was seen in the EI spectrum of this
derivative. Other ions presumably arise from fragmentation directed by
the double bonds at C-4 and C-10. Ions due to cleavage between C-6 and
C-7, at m/z 257 (
-fragment) and 200 (rearrangement ion
of
-fragment with transfer of a TMS group to the carboxylate
carbonyl), and between C-8 and C-9, m/z 273 (
-fragment)
are seen. This spectrum is consistent with the glucuronide at C-7.
GC-MS Analysis: Trihydroxy C18 Fatty Acids
CF/FAB-MS analyses of patients' urines show that the
ion m/z 505 is often associated with m/z 489,
suggesting that monounsaturated trihydroxy C18 fatty acids also occur.
PM
CI/GC-MS analysis of the PM derivatives of the
intact glucuronides support this observation with the expected (M
+ NH
)
ion, m/z 622, observed
in the spectra of numerous compounds. These CI spectra also contain
ions at m/z 268 and 204, characteristic of a glucuronide. Also
observed are fragment ions arising from the aglycone at m/z 355 (aglycone). The EI spectra of these compounds show many ions
arising from the glucuronic acid component.
PM-Methanolysis-TMS
The CI spectra of at least
eleven compounds show ions at m/z 445 (MH
)
and 462 ((M + NH
)
). The EI spectrum
of the major compound shows an intense ion at m/z 259 and ions
at m/z 303 and 213 consistent with a
9,10,?-trihydroxyoctadecenoic acid. However, many of the compounds
observed provide CI spectra that exhibit only weak ions at m/z 462 ((M + NH
)
). Their EI spectra
contain many of the ions that are seen in the spectra obtained from the
dihydroxy fatty acids, in particular m/z 173 and 259,
indicating
- and
-end fragments, respectively (see Fig. 5and Fig. 6). Although there is evidence of at least
one saturated trihydroxy fatty acid in the urine from patients, there
is no indication of diunsaturated species. Further characterization of
this group of hydroxylated fatty acids is ongoing.
GC-MS Analysis: Glycine and Taurine
Conjugation of Dihydroxy Fatty Acids
There is some evidence to suggest that the glucuronic acid
conjugated dihydroxy monounsaturated fatty acids, may also be
conjugated at the carboxyl group with glycine or taurine. These species
would give ions by negative ion FAB-MS at m/z 546 and 596,
respectively. Such ions are observed and the ion at m/z 596 is
prominent in the spectra of some patients (Fig. 3).
Glucuronidase treatment of these compounds, isolation by HPLC and
FAB-MS analysis, gave ions consistent with the glycine and taurine
conjugated aglycone. Methyl esterification of the compound represented
by m/z 596 gave rise to an intense ion at m/z 610.
This is consistent with the presence of a single unesterified carboxyl
group on the glucuronic acid. The other on the fatty acid component is
presumably blocked by taurine.
DISCUSSION
We observed that the FAB-MS spectra obtained by analysis of
urine from children with generalized peroxisomal disorders show several
unusual ions which had not been described previously. These ions were
among the ten most intense ions in spectra from the majority of
children with these disorders but have thus far not been observed in
the ten most intense ions in spectra obtained from normal children or
children with cholestatic liver disease. Therefore we postulated that
these ions represented an accumulation of novel compounds in the urine
of patients with peroxisomal disorders and that characterization of
these compounds would enhance our understanding of the pathophysiology
of peroxisomal disease. Our preliminary results suggested analysis of
these compounds may be useful as a diagnostic test. Using CI and EI
GC-MS analyses we established the structure of many of these compounds
as glucuronic acid conjugated, dihydroxy and trihydroxy, C16 and C18
fatty acids. The major components were shown to be the glucuronic acid
conjugated, monounsaturated dihydroxy fatty acids,
12,13-dihydroxy-9-octadecenoic acid and 9,10-dihydroxy-12-octadecenoic
acid. These compounds correspond to the major (M - H)
ion at m/z 489, observed in the FAB-MS spectra shown in Fig. 3. Similarly (M - H)
ions observed
in the FAB-MS spectra at m/z 491 and 487 correspond to the
glucuronide conjugated saturated and diunsaturated C18 dihydroxy fatty
acids described. A significant group of compounds that elute early in
the GC program (Fig. 4, Peaks 1-4) were
identified as dihydroxy saturated, monounsaturated and diunsaturated
C16 fatty acids. The glucuronic acid conjugates of these compounds are
consistent with (M - H)
ions at m/z 463, 461, and 459 in the FAB-MS spectra. Numerous compounds
eluting later than the dihydroxy fatty acids (Fig. 3, Peaks
12-22) are tentatively identified as trihydroxy C18 fatty
acids. It is of interest that other ions in the FAB-MS spectra obtained
with extracts of patients' urines, may correspond to C12 and C14
hydroxylated fatty acids (e.g. m/z 433, 417, and 415).
Several mechanisms exist which may explain the formation of the
dihydroxy fatty acids. Dihydroxy C18 fatty acids have been described
previously as chemical hydrolysis products formed in the
characterization of epoxy fatty acids (22, 23) but
have also been shown to arise by autoxidation in
vitro(24) . It would be reasonable to suppose that
dihydroxy fatty acids could be formed from unsaturated fatty acids
during storage of samples. However, the presence of a glucuronide
moiety on one of the hydroxyl groups indicates that the compounds we
have observed were formed in vivo, since conjugation with
glucuronic acid implies hepatic processing. Epoxy fatty acids, which
can be precursors of fatty acids with vicinal hydroxyl groups, may be
derived from the diet (25, 26, 27) or
synthesized in vitro. The epoxygenase pathway appears to be a
major microsomal oxidation route for polyunsaturated fatty acids in a
number of
tissues(23, 28, 29, 30, 31, 32, 33) ;
however, nonenzymatic autoxidation probably also occurs in
vivo(29, 34) . Vicinal dihydroxy C18 fatty acids
have also been observed as the products of microsomal metabolism in
vitro(35, 36) . The major metabolites from
linoleic and linolenic acids were the same as compounds 6, 7, 10, and
11 identified in Fig. 3. 12,13-Dihydroxy-9,15-octadecadienoic
acid was also observed(35) .
We postulate that
12,13-dihydroxy-9-octadecenoic acid and 9,10-dihydroxy-12-octadecenoic
acid are derived from linoleic acid (18:2(n-6)), an abundant
fatty acid in the body and that the saturated analogue,
9,10-dihydroxyoctadecanoic acid, may be derived from oleic acid
(18:1(n-9)). Similarly 12,13-dihydroxy-6,9-octadecadienoic
acid may be derived from
-linolenic acid (18:3(n-6)),
whereas oxidation of
-linolenic (18:3(n-9)) may give rise
to 9,10-dihydroxy-12,15-octadecadienoic acid and
15,16-dihydroxy-9,12-octadecadienoic acid. The direct relationship
between the position of the hydroxyl groups in the characterized
compounds and the double bond position in the C18 unsaturated fatty
acids is supportive of this contention, as is the threo configuration
demonstrated in the 9,10-dihydroxyoctadecanoic acid. This configuration
must result from a cis double bond as found in the unsaturated fatty
acids described above.
The unsaturated fatty acids, oleic, linoleic,
and
-linolenic acids, are readily
-oxidized in the
mitochondria and do not appear to accumulate in peroxisomal
disorders(37, 38) . In contrast
-linolenic and
docosahexanoic acid are poorly oxidized in the
mitochondria(38) . Accumulation of these and other unsaturated
fatty acids may inhibit mitochondrial
-oxidation (39) possibly channeling the fatty acids into pathways leading
to hydroxy compounds. It is unknown whether the C16 and C18 dihydroxy
and/or epoxy fatty acids are predominantly
-oxidized in the
peroxisome, such that dysfunction of this organelle leads to their
accumulation. However, our findings are consistent with this theory.
-Oxidation of numerous compounds has been localized to the
peroxisome (for review, see van den Bosch et al.(40) and Brown et al.(41) ) with accumulation
of these compounds in peroxisomal
disease(4, 5, 6, 7, 8, 9, 10, 11, 12) .
It is of interest that only one patient with a well characterized
single enzyme defect in fatty acid
-oxidation has been observed to
have prominent m/z 489 and 505 ions in the urine by FAB-MS
analysis.
The major C16 fatty acids observed, in particular the
monounsaturated dihydroxy C16 fatty acids, are the expected
-oxidation products of the major C18 dihydroxy species described
above. Hence, 7,8-dihydroxy-10-hexadecenoic acid and
10,11-dihydroxy-7-hexadecenoic acid are the expected products from a
single cycle of the
-oxidation pathway from
9,10-dihydroxy-12-octadecenoic acid and 12,13-dihydroxy-9-octadecenoic
acid, respectively. This does not preclude that
-oxidation has
occurred prior to hydroxylation. Hence 7,8-dihydroxy-10-hexadecenoic
acid could be derived from 16:2(n-6) although unsaturated C16
fatty acids do not appear to accumulate in generalized peroxisomal
disorders. Our findings suggest that the dihydroxy and/or epoxy fatty
acids are
-oxidized to a limited extent in these patients. It is
also conceivable that secondary inhibition of mitochondrial
-oxidation (42) may account for the accumulation of these
compounds.
Hydroxylation at the
or
-1 position or
hydroxylation allylic to the double bond and vicinal to an epoxide
would give rise to a trihydroxy species (30, 43, 44) and may channel through a
hydroperoxy-epoxy octadecenoate intermediate, which has been reported
as an autoxidation product of linoleic acid(45) .
At this
stage it is premature to suggest the mechanism by which the dihydroxy
and trihydroxy fatty acids accumulate in generalized peroxisomal
diseases. Although defective
-oxidation may be the underlying
defect, it is also possible that disruption of enzyme compartments
and/or the peroxisomal scavenging system for free radicals may lead to
accumulation of reactive oxygen species and oxidative attack on cell
lipids. The peroxisomes are potent sources of hydrogen peroxide because
of their high concentration of oxidases (41) and generate
superoxide radicals(46, 47) . Mechanisms in
peroxisomes counteract the production of these reactive oxygen species
by enzymes catalyzing the conversion of the superoxide radical ion
(O
) to hydrogen peroxide and then to
H
O and O
(48) . Failure to form the
intact peroxisome in disorders of peroxisomal biogenesis may perturb
the balance of formation and catabolism of these reactive oxygen
species and lead to lipid peroxidation. We would expect that
autoxidation in vivo would lead to the formation of other
oxidized metabolites such as monohydroxy fatty acids. These have not
been detected in our samples but such compounds have not been looked
for systematically. Finally other peroxisomal enzymes which are
involved in the conversion of unsaturated fatty acids to dihydroxy
fatty acids are also impaired (e.g. peroxisomal epoxide
hydrolase)(49) . The significance of this can not be assessed
at this point.
Polyunsaturated fatty acids in the phospholipids of
biological membranes are important in increasing membrane fluidity,
compressibility and permeability(21) . These in turn will
affect activity of membrane enzymes, cell-cell interaction, receptor
interactions and membrane transport. Accumulation of oxidized
metabolites of unsaturated fatty acids may therefore disrupt the normal
functioning of cells by this mechanism or by their own biological
activity. Further investigation of oxygenated fatty acids in normal
metabolism and peroxisomal disease will be useful in extending our
knowledge of the role of peroxisomes in fatty acid metabolism and of
the pathophysiology of peroxisomal disease.