RP-HPLC-fluorescence analysis of aliphatic aldehydes: Application to aldehyde-generating enzymes HACL1 and SGPL1

Article abstract of DOI:10.1194/jlr.D044230

Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RPHPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates. Copyright

Full text of DOI:10.1194/jlr.D044230

methods
RP-HPLC-fluorescence analysis of aliphatic aldehydes:
application to aldehyde-generating enzymes HACL1
and SGPL1
Serena Mezzar, Evelyn de Schryver, and Paul P. Van Veldhoven1
Department Cellular and Molecular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium
Abstract Long-chain aldehydes are commonly produced in
various processes, such as peroxisomal ␣-oxidation of long-
chain 3-methyl-branched and 2-hydroxy fatty acids and mi-
crosomal breakdown of phosphorylated sphingoid bases.
The enzymes involved in the aldehyde-generating steps of
these processes are 2-hydroxyacyl-CoA lyase (HACL1) and
sphingosine-1-phosphate lyase (SGPL1), respectively. In the
present work, nonradioactive assays for these enzymes were
developed employing the Hantzsch reaction. Tridecanal
(C13-al) and heptadecanal (C17-al) were selected as model
compounds and cyclohexane-1,3-dione as 1,3-diketone, and
the fluorescent derivatives were analyzed by reversed phase
(RP)-HPLC. Assay mixture composition, as well as pH and
heating, were optimized for C13-al and C17-al. Under opti-
mized conditions, these aldehydes could be quantified in
picomolar range and different long-chain aldehyde deriva-
tives were well resolved with a linear gradient elution by RP-
HPLC. Aldehydes generated by recombinant enzymes could
easily be detected via this method. Moreover, the assay al-
lowed to document activity or deficiency in tissue homoge-
nates and fibroblast lysates without an extraction step. In
conclusion, a simple, quick, and cheap assay for the study of
HACL1 and SGPL1 activities was developed, without rely-
ing on expensive mass spectrometric detectors or radio-
active substrates.—Mezzar, S., E. de Schryver, and P. P. Van
Veldhoven. RP-HPLC-fluorescence analysis of aliphatic al-
dehydes: application to aldehyde-generating enzymes HACL1
and SGPL1. J. Lipid Res. 2014. 55: 573–582.
Long-chain aliphatic aldehydes are produced during
various metabolic processes, such as microsomal oxida-
tion of long-chain alcohols, lysosomal degradation of
prenylated proteins, peroxisomal ␣-oxidation of 3-methyl-
branched fatty acids and 2-hydroxy long-chain fatty acids,
microsomal breakdown of phosphorylated sphingoid
bases, attack of plasmalogens by myeloperoxidase (MPO)-
derived hypochlorous acid, and microsomal degradation
of (lyso)plasmalogens (see Fig. 1A). The involved en-
zymes are fatty aldehyde dehydrogenase (ALDH3A2) (1, 2),
prenylcysteine oxidase 1 (PCYOX1) (3, 4), 2-hydroxyacyl-
CoA lyase (HACL1) (5, 6), sphingosine-1-phosphate lyase
(SGPL1) (7–9), MPO (10), and lysoplasmalogenase (11,
12). In particular, two of these enzymes, HACL1 and
SGPL1, have intensively been studied in our laboratory.
Their activity measurements are complicated by their low
specific activity [ف115 (5) and ف15 mU/g rat liver (13),
respectively], low Km (10–15 ␮M), and the considerable
interference of enzymes acting on their substrates (acyl-
CoA hydrolases for HACL1; phosphatases for SGPL1)
or on their cofactors (phosphatases acting on TPP, cofac-
tor for HACL1, and on pyridoxal-phosphate, cofactor for
SGPL1). In addition, the generated aldehyde is chemi-
cally not stable and subject to further metabolism, and
compounds normally used to trap the aldehyde or block
its metabolism interfere with the cofactor (pyridoxal-
phosphate). Moreover, the presence of plasmalogens in
mammalian tissues, which give rise to aldehydes under
acidic conditions (14), can cause a high background when
using nonradioactive substrates, especially in nervous
tissue.
Supplementary key words ␣-oxidation • Hantzsch reaction • peroxi-
somes • phytanic acid • sphingosine-1-phosphate
Aldehydes are highly susceptible to both enzymatic and
chemical degradation. On the one hand, the high reactiv-
ity of aldehydes makes their detection in tissue homoge-
nates rather tricky; on the other hand, it can be exploited
to develop assays for their detection.
To measure HACL1 and SGPL1 activities in tissue or
cell lysates, various protocols were developed by our group.
Abbreviations: ALDH3A2, fatty aldehyde dehydrogenase; CHD,
cyclohexane-1,3-dione; Cn-al, saturated straight chain aldehyde con-
taining n carbon atoms; Cn-sphingosine, sphingosine analogue containing
n carbon atoms; HACL1, 2-hydroxyacyl-CoA lyase; IS, internal standard;
MPO, myeloperoxidase; OT, oxythiamine; PCYOX1, prenylcysteine
oxidase 1; RP-HPLC, reversed-phase HPLC; SGPL1, sphingosine-1-
phosphate lyase; TPP, thiamine pyrophosphate.
This work was supported by grants from the Flemish “Fonds voor Wetenschap-
pelijk onderzoek” (G.0581.09N; G.0721.10N) and the Katholieke Universiteit
Leuven (OT/08/040). S.M. was supported in part by ELA no. 2010-00215
and OT/08/040, both granted to Dr. M. Baes.
Manuscript received 18 September 2013 and in revised form 4 December 2013.
Published, JLR Papers in Press, December 9, 2013
DOI 10.1194/jlr.D044230
1 To whom correspondence should be addressed.
e-mail: paul.vanveldhoven@med.kuleuven.be
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 55, 2014
573

Fig. 1. Long-chain aliphatic aldehydes are produced during various metabolic processes. A: Schematic representation of aldehyde pro-
duction in mammals during various enzymatic reactions, such as microsomal oxidation of long-chain alcohols, lysosomal degradation of
prenylated proteins, peroxisomal ␣-oxidation of 3-methyl-branched fatty acids and 2-hydroxy long-chain fatty acids, attack of plasmalogens
by MPO-derived hypochlorous acid, and microsomal breakdown of phosphorylated sphingoid bases. Double black arrows point to multiple
steps. B: Scheme for the Hantzsch reaction.
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Journal of Lipid Research Volume 55, 2014

Initially, home-made radioactive substrates were used,
such as 2-hydroxy-3-methyl-[1-14C]hexadecanoyl-CoA (5)
or 2-hydroxy-[1-14C]octadecanoyl-CoA (6), converted
by HACL1 into [1-14C]formyl-CoA and 2-methylpentade-
canal or heptadecanal, and [4,5-3H]D,e-sphinganine-
1-phosphate (9), converted by SGPL1 into [2,3-3H]
hexadecanal and phosphoethanolamine. Recently, given the
issues concerning the use, synthesis, and waste disposal of
radioactive substrates, nonlabeled 2-hydroxyoctadecanoyl-
CoA was used as substrate for HACL1 (6), followed by
derivatization of the generated aldehyde to the corre-
sponding 2,4-dinitrophenylhydrazone, extraction, and
reversed-phase (RP)-HPLC analysis. In addition, Jansen
et al. (15) used 2-hydroxyphytanoyl-CoA as a substrate
for HACL1 and analyzed the produced pristanal as
an ethoxime derivative by GC-MS. Related to SGPL1,
other groups introduced alternative substrates which
give rise to a fluorescent aldehyde, either directly such
as ␻-NBD-sphingosine-1-phosphate (16) and ␻-BODIPY-
C13-sphingosine-1-phosphate (17), or via ␤-elimination
such as ␻-(2-oxo-2H-chromen-7-yloxy)-C5-sphingosine-1-
phosphate (18). Alternatively, SGPL1-produced aldehydes
were detected by mass spectrometry after suitable derivati-
zation with semicarbazide followed by LC-ESI (sphingosine-
1-phosphate as substrate) (19), pentafluorobenzyloxime
followed by GC-EI (C17-sphinganine-1-phosphate as sub-
strate) (20), or 2-diphenylacetyl-1,3-indandione-1-hydrazone
followed by LC-MS/MS (sphingosine-1-phosphate as
substrate) (21).
Recombinant enzymes
Human HACL1 was expressed in Saccharomyces cerevisiae
CB80 strain (MAT␣, ura3-52, leu2-1, trp1-63, his3-200)
transformed with pPF17, coding for an N-His6-tagged fusion
(26). Human SGPL1 was expressed in Escherichia coli
Top10F’ cells transformed with plasmid pVB001, coding for
an N-His6-tagged fusion (7).
Synthesis of lipids
Saturated long-chain aldehydes were commercially ob-
tained or synthesized in a similar way as described previously
for C17-al (5); 2-hexadecenal was obtained by periodate
treatment of D,erythro-sphingosine (27). Purity of alde-
hydes was verified by GC-MS and/or derivatization with
2,4-dinitrophenylhydrazine (6). 2-Hydroxyoctadecanoyl-CoA
was prepared as described before (6). D,erythro-sphingosine,
D,erythro-sphinganine-1-phosphate, and 1-O-1’-(Z)-octade-
cenyl-2-oleoyl-sn-glycerol-3-phosphocholine were obtained
from Avanti Polar Lipids. Decahydroacridine derivatives
of C13-al and C17-al were prepared by mixing the alde-
hydes (100 nmol dissolved in 50 ␮l methanol) with 50 ␮l
derivatization mixture and 50 ␮l water (see below). The
reaction mixtures were loaded on an activated Bond Elut
C18 cartridge (100 mg; Varian), and the derivatives were
eluted with methanol. After drying, residues were reconsti-
tuted in 80% (v/v) methanol containing 10 mM ammo-
nium acetate (ف80 ␮M), and an aliquot was injected at
10 ␮l/min on line in a 1100 Series LC/MSD Trap-VL
(Agilent) equipped with ESI (nebulizer 15 psi, dry gas
5 l/min, dry temperature 325°C) and scanned for m/z
100–650 for 2 min.
In the present work, we investigated the Hantzsch reac-
tion for carbonyl compounds in order to allow the use of
nonlabeled substrates and an inexpensive derivatization
reagent (see Fig. 1B). Tridecanal (C13-al) and heptadeca-
nal (C17-al) were employed as model compounds and
cyclohexane-1,3-dione (CHD) as 1,3-diketone, followed by
RP-HPLC of the fluorescent decahydroacridine deriva-
tives. The first application of CHD to measure aldehydes,
in casu formaldehyde, was described by Sawicki and Carnes
in 1968 (22).
Enzyme measurements
The substrate 2-hydroxyoctadecanoyl-CoA or sphinganine-
1-phosphate was incubated at 37°C with purified recombi-
nant enzyme, tissue homogenate, or cell lysate in baked
Pyrex 5 ml glass tubes. Reaction mixtures for activity measure-
ment consisted of 50 mM Tris-HCl (pH 7.5), 0.8 mM MgCl2,
20 ␮M TPP, 6.6 ␮M BSA, and 40 ␮M 2-hydroxyoctade-
canoyl-CoA for HACL1 (6); and 0.1 M K-phosphate buffer
(pH 7.5), 25 mM NaF, 0.1 mM Na-orthovanadate, 0.25 mM
pyridoxal-phosphate, 1 mM DTT, 1 mM EDTA, 0.1% (v/v)
Triton X-100, and 40 ␮M sphinganine-1-phosphate for
SGPL1 (8). Reactions for tissue homogenates and cell
lysates were halted after 5 min (HACL1) or 60 min (SGPL1)
by adding 1 vol of methanol (see below).
EXPERIMENTAL PROCEDURES
Animals and cells
Animal studies were approved by the University Eth-
ics Committee, and all animal experiments were per-
formed in accordance with the institutional and national
guidelines for care and use of laboratory animals. Swiss
Webster mice were kept under conventional conditions
in the animal housing facility of the KU Leuven, with a
12 h light and dark cycle, and fed a regular chow diet.
At the time of the experiment, mice were anesthetized
with an overdose of Nembutal (approximately 150 ␮g/g
body weight), and tissues were dissected. Human fibro-
blasts (23) and murine fibroblasts, derived from C57BL6
or Sgpl1Ϫ/Ϫ mice (24, 25), were cultured as described in
the cited references.
Acidic pretreatment of tissue homogenates
Mouse livers and brains were homogenized (1 part/20 vol)
in isotonic buffer [0.25 M sucrose, 5 mM MOPS (pH 7.2),
0.1% (v/v) ethanol], and 40 ␮l homogenates were ex-
posed to either 0.3 M HCl or NaCl for 15 min at 65°C.
Subsequently, the acid-treated samples were neutralized
with NaOH. The generated aldehydes were then analyzed
by RP-HPLC.
RP-HPLC-fluorescence analysis of aliphatic aldehydes
575

RP-HPLC analysis of aldehydes
dimethylsulfoxide, tetrahydrofuran, acetonitrile, and
dimethylformamide, were also stimulatory but not to
the same extent (ethanol, dimethylsulfoxide), or they
displayed a sharp concentration optimum (data not shown).
For shorter aldehydes, e.g., C13-al, the requirement for
methanol was less stringent (Fig. 2F). From the data ob-
tained, we evaluated the best assay conditions, which al-
lowed for the quantification of both C13-al and C17-al.
The selected parameters, based on three subsequent
rounds of optimizations on standards, were 1.2 M am-
monium acetate, 33 mM CHD, 14% (v/v) acetic acid,
33% (v/v) methanol, and heating at 65°C for 60 min.
Other factors, which could cause interference when
analyzing biological samples, were considered, such as
plasmalogen breakdown at low pH or high temperature
(see below) or peroxidation of polyenoic acids by ex-
treme heat; hence, 65°C instead of 90°C was chosen
(see also Ref. 28). The selected conditions allowed for
the quantification of C17-al in picomolar range (low
sensitivity, 10–150 pmol; medium sensitivity, 1–10 pmol)
(Fig. 3A). LC-ESI-MS analysis of the C13-al and C17-al
derivatives revealed a compound with m/z 386.2 for
C13-al (Fig. 3B) and m/z 442.2 for C17-al (data not
shown), which upon fragmentation, yielded a fragment
of m/z 217.9, corresponding to the decahydroacridine
moiety and confirming the postulated structures (Fig. 3C).
Subsequently, we checked to which extent aldehydes
with rather similar chain length could be discriminated:
a good resolution of different long-chain aldehydes was
obtained, and the elution time related to the number of
carbon atoms (Fig. 4).
According to our optimized protocol, assay mixtures
[100–200 ␮l spiked with internal standard (IS, 0.5–2.0 nmol)]
were mixed with 1 vol of ice-cold HPLC-grade methanol,
followed by 1 vol derivatization medium [3.6 M ammo-
nium acetate, 100 mM CHD, 42% (v/v) acetic acid], cap-
ping and heating for 60 min at 65°C. Upon cooling, 2 vol of
methanol, relative to the initial sample volume, were
added to reach a methanol concentration of 60%. The lat-
ter step was omitted when optimizing the derivatization of
aldehyde standards. After a 1000 g spin, the supernatant
was transferred to inserts of HPLC-vials (Alltech) and 1/50
up to 1/5 of the total volume was injected on a Symmetry®
C18 column (4.6 × 150 mm; 5 ␮m; 100 Å; Waters) equili-
brated with 80% (v/v) methanol. Bound derivatives were
eluted with a gradient (80–100% methanol) over 12 min
and detected by fluorimetry (Waters 2475 Multi-Wavelength
Fluorescence Detector; Ex 390 nm; Em 460 nm; emission
mode; EUFS 3000 or lower). Wavelength settings were
based on Ex/Em spectra obtained in Aminco SPF500 fluo-
rimeter for the decahydroacridine derivatives of formalde-
hyde and tetradecanal (C14-al) (data not shown).
RESULTS AND DISCUSSION
Optimization of assay conditions
To date, the longest aldehyde that has been analyzed
via the Hantzsch reaction using CHD as 1,3-diketone
was decanal (C10-al) (28, 29). Based on this report, dif-
ferent concentrations of components, as well as pH and
heating parameters, were tested to optimize the detec-
tion of C13-al and C17-al (Fig. 2A–E). In addition, the
presence of methanol as a cosolvent, not investigated in
previous reports, was demonstrated to be essential for
the quantitative conversion of C17-al into its fluorescent
derivative (Fig. 2F). Other cosolvents, such as ethanol,
The presence of proteins can affect the recovery of
aliphatic aldehydes (e.g., Schiff base with amino groups,
hydrophobic interactions, trapping in pelleted proteins
or in interphase during extraction), and negative effects
of proteins have been observed also in CHD-based assays
containing no or less methanol [59% recovery for C6-al in
presence of 16.7 mg rat liver/ml assay mixture (28); 65–72%
Fig. 2. Optimization of aldehyde derivatization.
Concentration of the different assay components,
pH, and heating parameters were varied to optimize
the derivatization of C13-al (triangles) and C17-al
(open squares) (1.5 and 2 nmol, respectively). Fluo-
rescent derivatives were directly separated by
RP-HPLC, without dilution with methanol, and the
integrated area of the signal was plotted against the
variable parameter, while other parameters (1.3 M
ammonium acetate, 35.7 mM CHD, 13.3% (v/v)
acetic acid, 80°C temperature, 90 min heating time,
40% (v/v) methanol, based on initial tests) were
kept constant, unless otherwise mentioned. A: Ef-
fect of ammonium acetate. B: Effect of CHD. C: Ef-
fect of acetic acid. D: Effect of temperature (1.6 M
ammonium acetate, 14% (v/v) acetic acid, 60 min
heating time). E: Effect of heating time (1.6 M am-
monium acetate, 14% (v/v) acetic acid). F: Effect
of methanol (1.2 M ammonium acetate, 14% (v/v)
acetic acid, 65°C temperature, 60 min heating
time).
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Journal of Lipid Research Volume 55, 2014

Fig. 3. RP-HPLC and MS analyses of fluorescent derivatives. A: RP-HPLC analysis of the C17-al derivative.
Integrated fluorescence peak area, obtained with EUFS setting of the detector at 3,000, was plotted versus
amount of injected C17-al present in 10 ␮l of reaction mixture (600 ␮l in total). Insert shows analysis of lower
amounts of aldehyde at a higher sensitivity (EUFS 1000). B: Mass spectrum scan of the C13-al derivative
[positive mode, smoothed (0.2; 1; GA)]. Semi-purified derivatives were subjected to LC-ESI-MS, revealing a
major ion of m/z 386.2 for the C13-al derivative (calculated mass 385.3) and m/z 442.2 for the C17-al deriva-
tive (calculated mass 441.36) (not shown). Smaller peaks represent contaminants present in both spectra.
C: Fragmentation spectrum of the compound with m/z 386.2 (position indicated by diamond) with a charac-
teristic peak at m/z 217.9, corresponding to the aromatic ring structure.
RP-HPLC-fluorescence analysis of aliphatic aldehydes
577

Fig. 4. RP-separation of long-chain aldehydes according to chain length. Aldehydes (1-3 nmol, based on derivatization with 2,4-
dinitrophenylhydrazine) with different chain lengths (13–18 carbon atoms) were mixed, converted to CHD derivatives, separated by RP-
HPLC, and detected by fluorimetry (Ex 390 nm, Em 460 nm). The insert shows the relationship between elution time and aldehyde chain length.
The drop in fluorescence near 1.8 min is due to a transient closure of the emission filter to protect the detector against too high intensity.
biological samples, it is suggested to use less than 4 mg
tissue or less than 0.8 mg protein per 200 ␮l assay (see
Fig. 5D).
recovery for C7-al in presence of 50 ␮l human plasma/ml
assay mixture (30); chain length-dependent decrease of
recovery, down to 8% for C10-al, in presence of 0.33 ml
human plasma/ml assay mixture (29)]. Nonetheless, re-
covery of medium-chain aldehydes (C13-al) was excellent
under our conditions [33% (v/v) methanol during de-
rivatization] in presence of increasing amounts of tissue
homogenate. However, recovery of C17-al decreased (15% at
2 mg tissue/assay). Attempts to prevent this by adding de-
tergents or increasing the amount of cosolvent were not
successful (data not shown). During these trials, extra
methanol was added to the derivatized samples: this in-
creased the amount of C17-al signal considerably, with-
out (Fig. 5A) or with proteins (Fig. 5B). Hence, the
formed C17-al derivative is partially lost after cooling and
spinning, perhaps due to solubility problems, and this
can be counteracted by adding extra methanol after de-
rivatization, or in the opposite sense, worsened by adding
water. With this modification, recoveries of IS with brain
or liver homogenate were improved (Fig. 5C, D). More
importantly, the ratio of endogenous aldehydes over C17-al
(Fig. 5E) or C13-al (data not shown) was linear with the
amount of tissue analyzed, up to 16 mg tissue/assay, which
allows for reliable quantifications. When applying the
method for the analysis of enzyme-derived aldehydes in
Application to biological samples
First, we verified whether our optimized method was
suitable for the measurement of enzyme-produced alde-
hydes. As expected, incubation of recombinant human
HACL1 with 2-hydroxyoctadecanoyl-CoA resulted in the
formation of C17-al (Fig. 6A); similarly, the cleavage of
sphinganine-1-phosphate by recombinant human SGPL1
could be demonstrated (Fig. 6B).
Then, given our intention to measure HACL1 activity in
cells or tissues of patients or mice with peroxisomal disor-
ders (31), the possible interference of plasmalogens, which
are present in variable amounts in different tissues and
whose levels depend on functional peroxisomes, was veri-
fied. Indeed, the vinyl-ether bond of plasmalogens, pres-
ent at 1–10 ␮mol/g in some tissues (brain, sebaceous
glands), is sensitive to acidic conditions (14), resulting in
formation of aldehydes and, consequently, in a high back-
ground when analyzing enzymatically formed aldehydes.
Comparison of the fatty aldehyde profiles of liver or brain
samples with or without pretreatment with acid showed that
plasmalogen hydrolysis was minimal when derivatization
578
Journal of Lipid Research Volume 55, 2014

was performed under optimized conditions (Fig. 7A, B),
but it was increased at 90°C (data not shown). The acidic
pretreatment caused the expected generation of alde-
hydes, especially hexadecanal (C16-al) and octadecanal
(C18-al) in brain (Fig. 7B). Through derivatization of
synthetic plasmalogen (1-O-1’-(Z)-octadecenyl-2-oleoyl-sn-
glycero-3-phosphocholine), the amount of hydrolysis was
estimated at 6.2% (Fig. 7C). Hence, the peaks seen in the
nontreated conditions likely represent both free fatty alde-
hydes (32) and plasmalogen-derived aldehydes. This
might, depending on tissue/cell type investigated and on
the aldehyde produced, call for correction by appropriate
blank incubations (see Fig. 6D, F). Compared with the
classical derivatization with DNPH, the CHD method is
less aggressive. In fact, derivatization with CHD is per-
formed at pH ف5.5, while derivatization with DNPH, be-
ing only soluble at low pH, is generally carried out under
acidic conditions (e.g., 1–2 N HCl or H2SO4). Hence, per-
oxidation of polyenoic acids, resulting in formation of sec-
ondary products, such as 2,4-hexadienal, is less with CHD,
as was also pointed out by Yoshino et al. (28). Moreover, as
shown in this work, plasmalogen hydrolysis is limited. Fur-
thermore, DNPH-based measurements rely on a photo-
metric detector, sensitivity being determined by its ␧ value
(23,000–28,700; solvent dependent), whereas CHD relies
on a fluorimetric detector (lowest limit at highest sensitiv-
ity, approximately 5 fmol). In addition, CHD is preferred
over DNPH, as the latter is classified as a dangerous chemi-
cal (risk class E4), whereas CHD is considered a safe chem-
ical (at least in the European Union). Moreover, CHD is
much cheaper than dansylhydrazine or other (even more
expensive) fluorescent hydrazides (33–37) that have been
used to analyze (short-chain) fatty aldehydes via HPLC.
Like DNPH, these compounds react optimally under
acidic conditions.
Subsequently, we measured HACL1 activity in mouse
liver homogenates (Fig. 6C). A gender dependency, so far
not reported, was found (106 12 mU/g liver in females,
n = 4, and 71 19 mU/g liver in males, n = 6). The values
obtained were comparable to those previously measured
in rodent liver homogenates using long-chain 2-hydroxyacyl-
CoA as substrate (5, 6, 23). Linearity of HACL1 activity
in mouse liver homogenates is limited to 5 min (at 1 mg
Fig. 5. Recovery of long-chain aldehydes in presence of proteins.
A: Effect of methanol on standards. C13-al (triangles) and C17-al
(open squares) (1.5 and 2 nmol, respectively) were derivatized fol-
lowing the optimized protocol in the presence of 33% (v/v) metha-
nol. Upon derivatization, equal volumes of water, water/methanol,
or pure methanol were added to the assay to obtain the indicated
methanol concentration. After centrifugation, the supernatant was
subjected to RP-HPLC, and the fluorescent derivatives were de-
tected by fluorimetry (Ex 390 nm, Em 460 nm). Values represent
means of two determinations. B: Effect of methanol on standards
in presence of proteins. Brain homogenates (2 mg tissue) were
spiked with C13-al (triangles) and C17-al (open squares) before
derivatization. Upon derivatization, mixtures were processed as de-
scribed for (A). Integrated peak areas of C13-al and C17-al deriva-
tives are plotted versus final methanol concentration. C. Recovery
of IS in presence of proteins. Increasing amounts of brain and liver
homogenates were spiked with C13-al and C17-al before derivatiza-
tion. Water/methanol or methanol was added to the derivatized
sample in order to have a final concentration of either 30% or 60%
(v/v). Derivatives were separated by RP-HPLC. Recoveries of C13-al
and C17-al derivatives were plotted versus amount of tissue per as-
say. Open triangles: C13-al, 60% methanol; filled triangles: C13-al,
30% methanol; open squares: C17-al, 60% methanol; filled squares:
C17-al, 30% methanol. D. Recovery of IS in presence of proteins.
Increasing amounts of brain and liver homogenates were spiked
with C13-al (triangles) and C17-al (open squares) before derivatiza-
tion. Methanol was added to the derivatized sample to a final con-
centration of 60% (v/v). Derivatives were separated by RP-HPLC.
Integrated peak areas of C13-al and C17-al derivatives were plotted
versus amount of tissue per assay. E. Analysis of endogenous alde-
hydes. Increasing amounts of brain and liver homogenates were
spiked with C17-al before derivatization and separation of the fluo-
rescent derivatives by RP-HPLC. The ratio of endogenous C16-al
(squares) and C18-al (open triangles) over C17-al standard was
plotted versus amount of tissue per assay.
RP-HPLC-fluorescence analysis of aliphatic aldehydes
579

Fig. 6. Analysis of enzymes producing fatty aldehydes. A: Human recombinant HACL1 (5 ␮g purified protein) was incubated at 37°C for
5 min with 2-hydroxyoctadecanoyl-CoA as substrate (40 ␮M). A blank reaction was also included, where no substrate was added. The reac-
tion mixtures were fortified with IS (C13-al; 1.5 nmol) before derivatization. B: Human recombinant SGPL1 (0.8 mg protein of soluble E.
coli extract) was incubated at 37°C for 60 min with sphinganine-1-phosphate as substrate (40 ␮M). A blank reaction was also included,
where no substrate was added. The reaction mixtures were fortified with IS (C17-al; 2 nmol) before derivatization. C: Liver homogenates
of Swiss mice (1 mg tissue, corresponding to ف0.2 mg protein) were incubated at 37°C for 5 min with 2-hydroxyoctadecanoyl-CoA (40 ␮M),
mixtures were spiked with IS (C13-al; 0.74 nmol), and formed aldehydes were analyzed as described in (A). D: Liver homogenates of Swiss
mice (2 mg tissue, corresponding to ف0.4 mg protein) were incubated at 37°C for 60 min with sphinganine-1-phosphate (40 ␮M), and
formed aldehydes were analyzed as described in (B). E: Human fibroblasts were grown either in standard medium (Ct) or in the presence
of 1 mM oxythiamine (OT) for two days before HACL1 activity measurement. Fibroblast lysates (0.2–0.5 mg protein) were then incubated
at 37°C for 5 min with 2-hydroxyoctadecanoyl-CoA (40 ␮M). Blank reactions were also included, where no substrate was added. The reac-
tion mixtures were spiked with IS (C13-al; 0.74 nmol) before derivatization. Note the presence in fibroblasts of endogenous C16-al and
C18-al, eluting at 7.4 and 8.7 min, respectively. F: Lysates of mouse fibroblasts (ف0.2 mg protein), obtained from wild-type or Sgpl1Ϫ/Ϫ
C57BL6 embryos, were incubated at 37°C for 60 min with sphinganine-1-phosphate (40 ␮M). Blank reactions were also included, where no
substrate was added. The reaction mixtures were spiked with IS (C17-al; 2 nmol) before derivatization, followed by analysis of the generated
C16-al. Assays without substrate serve to correct for nonSGPL1-derived C16-al.
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Journal of Lipid Research Volume 55, 2014

tissue/assay), while activity of the recombinant enzyme was
linear at least up to 30 min. This difference can be ex-
plained by the presence of a high acyl-CoA hydrolase activity
in mouse liver homogenates (approximately 1.8 U/g mouse
liver under the HACL1 assay conditions), responsible for the
degradation of the substrate during incubation. SGPL1 activ-
ity could easily be measured in mouse liver homogenates
(4.2 1.9 mU/g liver in females, n = 3; 3.4 0.4 mU/g
liver in males, n = 3) (Fig. 6D). In agreement with previous
reports (13), SGPL1 activity in liver homogenates (<4 mg
tissue/assay) was linear up to 1 h of incubation.
Of clinical importance, lyase activities could also be
measured in human fibroblast lysates [187 29 pmol/
min/mg protein for HACL1 (n = 4), and 19 pmol/min/
mg protein for SGPL1 (n = 2)]. Hence, the assay can be
employed to study enzyme deficiencies in patients. To sim-
ulate HACL1 deficiency, so far not yet reported, HACL1
activity was measured in human fibroblasts exposed to
oxythiamine (OT), which blocks the coenzyme functions
of thiamine. In fact, OT-diphosphate competes with TPP
for binding to apo-enzymes (23). As reported before, OT
exposure resulted in inhibition of ␣-oxidation (23) and re-
duced HACL1 activity (Fig. 6E). Likewise, SGPL1 deficiency
in man has not been diagnosed, but a clear drop in aldehyde
production was seen in Sgpl1Ϫ/Ϫ mouse fibroblasts (Fig. 6F).
Note that the use of sphingosine-1-phosphate as SGPL1
substrate is not recommended for this assay. The recovery
of the produced 2-hexadecenal from homogenates
is not complete (unpublished data), as was also noticed by
Berdyshev et al. (19). Furthermore, the Hantzsch reaction
with synthetic 2-hexadecenal does not proceed well, giving
rise to spurious peaks, whereas derivatization with 2,4-
dinitrophenylhydrazine works smoothly (unpublished data).
Both observations are likely due to the reactivity of the
␣,␤-unsaturated carbonyl group toward sulphydryl and
amino compounds. Adduct formation of ␣,␤-unsaturated
aldehydes with proteins and DNA has long been known
(38), and more recently, it has also been demonstrated for
the lyase metabolite of sphingosine-1-phosphate (39, 40).
In conclusion, a simple and cheap assay for aliphatic al-
dehydes is described, without an extraction step or need
for a mass spectrometric detector, and it is generally ap-
plicable to enzymes producing aldehydes, as demonstrated
for HACL1 and SGPL1. For quantitative measurements,
an appropriate IS has to be selected, depending on the
chain length of the produced aldehyde and the endoge-
nous fatty aldehydes present in tissue/cells investigated.
Likewise, the assay can be adapted to other analytical tech-
niques in which aldehydes are quantified, such as plasmal-
ogen content/composition analyses.
Fig. 7. Plasmalogen cleavage. Liver homogenates (ف2 mg tissue) (A),
brain homogenates (ف2 mg tissue) (B), and 1-O-1’-(Z)-octadecenyl-
2-oleoyl-sn-glycero-3-phosphocholine (2 nmol) (C) were pretreated
with either HCl or NaCl for 15 min at 65°C before aldehyde derivati-
zation and separation of the fluorescent derivatives by RP-HPLC.
Also a blank, containing no tissue or lipid, was analyzed. C16-al and
C18-al eluted at 7.4 and 8.8 min, respectively. The drop of the blue
line at 7.4 min and 8.8 min in (B) is due to a transient closure of the
emission filter to protect the detector against too high intensity. The
peak at 7.4 min in (C) is likely due to the presence of a contaminant
The authors appreciate the help of S. Asselberghs (KU Leuven,
LIPIT) with the MS analysis and the interest of Dr. M. Baes
(Laboratory for Cell Metabolism, Dept. Pharmaceutical and
Pharmacological Sciences, KU Leuven) in our work.
in the stock solution of 1-O-1’-(Z)-octadecenyl-2-oleoyl-sn-glycero-3-
phosphocholine.
RP-HPLC-fluorescence analysis of aliphatic aldehydes
581

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