Supplemental Data
CHEMICALS AND REAGENTS
3,3-dimethylglutaric, methoxyamine hydrochloride, sodium chloride, pyridine and
acetyl chloride were from Sigma Aldrich. N,O-bis(trimethylsilyl)trifluoroacetamide
containing 1% trimethylchlorosilane was from Regis Technologies. Ethoxyamine
hydrochloride was from Fluka and ethyl acetate, iso-octane, 25% ammonia and nbutanol were from Merck.
URINE ORGANIC ACID ANALYSIS
Creatinine was used to correct for urine dilution and samples were diluted to a
creatinine concentration of 1 mmol/L. Diluted urine samples (1 mL) were mixed with
100 L of 1 mol/L methoxyamine hydrochloride containing 1 mmol/L 3,3dimethylglutaric acid (internal standard) and incubated at 60oC for 30 minutes. Fifty
L of 6 mol/L hydrochloric acid was added, followed by solid sodium chloride and
solvent extraction with 5 mL ethyl acetate. The ethyl acetate phase was removed
and 10 L of 25% ammonia was added to minimize loss of volatile organic acids.
Ethyl acetate was removed by evaporation under an air stream and 20 L of pyridine
and 80 L of N,O-bis(trimethylsilyl)trifluoroacetamide containing 1%
trimethylchlorosilane was added, the mixture incubated at 80oC for 30 minutes,
cooled and 900 L of iso-octane added. This process is referred to as “standard
derivatization”.
Experiments were performed in which alternative derivatives were prepared and
analysed by GC-MS to assist in the structural characterization of some of the novel
organic acids. GC-MS conditions were identical. Control urine was analysed in each
experiment and the chromatograms were checked for: a) presence of a new
derivative and absence of the standard derivative in affected samples using the
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alternative derivatization, b) absence of the new derivative in the control sample
using the alternative derivatization, c) absence of the new derivative in affected
samples using standard derivatization.
Ethoxyamime/trimethylsilyl derivatives were prepared by substitution of ethoxyamine
hydrochloride for methoxyamine hydrochloride in the above analytical process. All
other conditions were the same. Butyl/trimethylsilyl derivatives were prepared by
heating the residue after urine solvent extraction with n-butanol:acetyl chloride (9:1
v/v) for 30 minutes at 65oC. Excess reagents were then removed under an air stream
and the residue trimethylsilylated as described above.
GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Two GC-MS systems were used to analyse the derivatized extracts. System 1 was
an Agilent 5973 GC-MS system fitted with a 30 m HP-5MS column, internal diameter
0.2 mm, film thickness 0.25 m. One µL of derivatized extract was injected using
splitless injection, the column was held at 80oC for 1 minute followed by a
temperature programmed ramp of 8oC/minute to 300oC. The mass spectrometer was
scanned from 50 to 550 m/z every second. Semi-quantitation of organic acids was
performed assuming that TIC peak areas gave the same molar response as the
internal standard. System 2 was a Perkin Elmer Turbomass GC-MS fitted with a
J&W 20 m BP5-MS column, internal diameter 0.18 mm, film thickness 0.18 m.
SYNTHESIS OF METABOLITES
Metabolites B, C, E and G were prepared from commercially available 4-hydroxy-6methyl-2-pyrone (D) by the following reactions: Catalytic hydrogenation of D in
ethanol solution with 10% palladium on carbon afforded 3,5-dihydroxyhexanoic 1,5
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lactone (B) (Huck et al. 2001). Oxidation of B with Jones reagent gave 5-hydroxy-3ketohexanoic 1,5-lactone (J). The methoxyamine derivative of J afforded two peaks
by GC-MS analysis with identical mass spectra (54 (100%), 59 (68%), 68 (38%), 113
(29%), 157 (M+, 8%)). Hydrolysis of J with 1 mol/L sodium hydroxide yielded two
peaks of 5-hydroxy-3-ketohexanoic acid (E) with identical mass spectra due to oxime
syn/anti isomerism). Dehydration of B with phosphorous pentoxide in diethyl ether
gave 6-methyl-5,6-dihydro-2-pyrone (K). Hydrolysis of K with 1 mol/L sodium
hydroxide yielded cis-5-hydroxyhex-2-enoic acid (C). Hydrolysis of B with 1 mol/L
sodium hydroxide gave metabolite G.
IDENTIFICATION OF ORGANIC ACIDS
Peaks B, C, D, E and G in patient samples were identified as the
methoxyamine/TMS derivatives of 3,5-dihydroxyhexanoic 1,5 lactone, cis-5hydroxyhex-2-enoic acid, 4-hydroxy-6-methyl-2-pyrone, 5-hydroxy-3-ketohexanoic
acid and 3,5-dihydroxyhexanoic acid respectively by spectral comparisons with the
authentic compounds and co-elution of the endogenous and authentic compounds.
The spectra are shown in Fig. 1. Double chromatographic peaks for 3,5dihydroxyhexanoic 1,5 lactone and 3,5-dihydroxyhexanoic acid were observed in the
subject urine samples. Both compounds contain two chiral centers and this
observation indicates the formation of diastereoisomeric pairs in vivo. Single
chromatographic peaks were obtained for the synthetic compounds indicating
stereo-specificity in the synthesis. Synthetic derivatized 3,5-dihydroxyhexanoic 1,5
lactone corresponded to the slower eluting peak and synthetic derivatized 3,5dihydroxyhexanoic acid corresponded to the faster eluting peak of the pairs seen in
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subject urine samples. The remaining two compounds were provisionally identified
by comparisons with above compounds and the formation of alternative derivatives.
The spectrum of peak A, shown in Fig. 1A, has several features in common with that
of cis-5-hydroxyhex-2-enoic acid (Fig. 1C). M-15 ions are prominent in trimethylsilyl
derivatives (Halket 1993) and the ion of 259 m/z and the less abundant ion at 274
m/z indicated the same molecular weight of 274 Da. This molecular weight and the
abundant ion at 143 m/z, from cleavage α to a trimethylsilyl group (Rontani et al.
2008), suggested a 3-hydroxy unsaturated hexanoic acid. Formation of a
butyl/trimethylsilyl derivative produced a mass spectrum (73(100%), 143(81%),
157(26%), 159(28%), 243(14%)) consistent with this structure. Because of the interrelationship with 4-hydroxy-6-methyl-2-pyrone we propose a trans-configuration
about the double bond and provisionally identify peak A as trans-3-hydroxyhex-4enoic acid.
The spectrum of peak F, shown in Fig. 1F, has several features in common with that
of 5-hydroxy-3-keto-hexanoic acid (Fig. 1E). The M-15 ion at 304 m/z indicated the
same molecular weight of 319 Da and the abundant ion at 233 m/z is typical of
trimethylsilyl derivatives of 3-hydroxy acids (Rontani et al. 2008). The odd molecular
weight, in combination with an M-31 ion at 288 m/z and an 89 m/z fragment
suggested a methoxyamine derivative of a keto acid. This was confirmed by the
formation of an ethoxyamine/trimethylsilyl derivative which gave a homologous
spectrum (73(100%), 103(11%), 147(13%), 189(10%), 233(16%), 288(1%),
318(3%)) with several 14 m/z shifts. On the basis of these results, peak F was
provisionally identified as 3-hydroxy-5-ketohexanoic acid.
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Peaks A and C were not detected when derivatized metabolite G was analysed as a
pure standard, indicating that the former two unsaturated endogenous compounds
did not arise from thermal dehydration of the latter in the gas chromatography
injection system. In contrast to 3,5-dihydroxyhexanoic acid 1,5 lactone, 5-hydroxy-3ketohexanoic acid 1,5 lactone (peak J) was not detected in any urine samples. The
methoxyamine of J was synthesized to confirm this.
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References
Halket JM. Derivatives for Gas Chromatography-Mass Spectrometry. In: Blau K, Halket JM, eds.
Handbook of Derivatives for Chromatography. 2 Ed. Chichester: John Wiley & Sons 1993:297-325.
Huck W-R, Burgi T, Mallat T, Baiker A (2001) Asymmetric hydrogenation of 4-hydroxy-6-methyl-2pyrone: role of acid-base interactions in the mechanism of enantiodifferentiation. Journal of
Catalysis 200171-80
Rontani JF, Aubert C (2008) Hydrogen and trimethylsilyl transfers during EI mass spectral
fragmentation of hydroxycarboxylic and oxocarboxylic acid trimethylsilyl derivatives. J Am Soc Mass
Spectrom 19(1):66-75
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Fig. 1 Mass spectra of peaks A to G as methoxyamine/trimethylsilyl (TMS)
derivatives. Chiral centers are indicated by asterisks. A, trans-3-hydroxyhex-4-enoic
acid (provisional identification); B, 3,5-dihydroxyhexanoic 1,5 lactone; C, cis-5hydroxyhex-2-enoic acid; D, 4-hydroxy-6-methyl-2-pyrone; E, 5-hydroxy-3ketohexanoic acid; F, 3-hydroxy-5-ketohexanoic acid (provisional identification); G,
3,5-dihydroxyhexanoic acid.
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Fig. 2 Proposed metabolic pathways for the formation of the novel metabolites
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Table 1 Clinical and mutation details of biochemically characterized individuals with HMCS2 deficiency (cohort A)
Biochemical measurements were performed during the first presentation. Reference ranges are indicated in brackets.
Allele 2
mutation
Familypatient
Clinical features at
presentation
Symptomatic
presentations1
Exon 1
deletion
Exon 1
deletion
A1-1
hypoglycemia,
hepatomegaly,
metabolic acidosis
8m; 23m
-
0.3
3.1
19
9m
194
0.8
0.1
2.3
17
A1:
Free fatty acids
mmol/L (<0.7)
Consanguinity
Current age (years)
-
Ethnic origin
D-3-hydroxybutyrate
mmol/L (<0.6)
Lactate mmol/L (0.5 –
2.2)
Allele 1
mutation
Ammonium µmol/L
(<50)
Family:
Lebanese
A1-2
Distantly related
A1-3
6m
29
5.0
-
-
10
A1-4
10m; 11m
51
0.8
0.2
0.7
8
52
0.7
0.6
2.5
9
A2:
p.L266S
p.I407T
Greek
(c.797T>C)
(c.1220T>C)
A3:
p.G388R
p.G388R
Egyptian
(c.1162G>A)
(c.1162G>A)
Consanguineous
A2-1
hypoglycemia
3y1m
A3-1
hypoglycemia,
metabolic acidosis,
hepatomegaly
6m
-
1.1
0.2
2.7
7
Asymptomatic
-
-
-
-
5
Normal
-
0.2
-
8
A3-2
A4:
p.G169D
p.R505Q
Indian
(c.506G>A)
(c.1514G>A)
A4-1
hypoglycemia
2y5m
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1
y=year, m=month
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