CLIN.CHEM. 22/8,1330-1338(1976)
Structure of Some Aliphatic Dicarboxylic Acids Found
in the Urine of an Infant with Congenital Lactic Acidosis
Sven Llndstedt, Karin Norberg, GOran Steen, and Elisabeth Wahi
Gas chromatography/massspectrometry was used to
identify
a seriesof acids inurine and serum from a child
who died 26 h after birth in severe metabolic acidosiswith
high lactateexcretion.c/s-5-Decene-1, 10-dioic acid and
cis-5-dodecene-1 , 12-dioic acid were synthesized and used
as references. The following acids were found: hexane1,6-dioic acid, octane-1,8-dioic acid, decane-1,10-dioic
acid, dodecane-1,12-dioic
acid, cis-5-decene-1,10-dioic
acid, cis-5-dodecene-.1, 12-dioic acid, cis-5-tetradecene- 1,1 4-dioic acid, trans-3-decene-1 , 10-dloic acid, and
trans-3-dodecene1,12-dioic acid. The concentration of
C6 to C14 acids in the patient’s urine was 3.7 mol/mol of
creatinine; it was <0.2 moi/mol of creatinine In eight
normal newborns and ‘-0.1 mol/mol of creatinine in a case
of fructose-i ,6-biphosphatase deficiency with lactic aci-
dosis. 5-cis-Dodecenediolc acid was present in highest
concentration:
1 mol/mol of creatinine in urine and 61
smol/lIter in serum. We propose that impaired /9-oxidation,
probably at the acyl-C0A-dehydrogenase
step, resulted
in the formation of the observed acids. The parents were
consanguineous, and a sibling died with the same clinical
picture, which suggests a genetic defect.
AdditIonal Keyphrases:
cls-5-decene- 1,1O-dioicacid #{149}
cis-5-cAcene- 1,12-dioic acid. #{149}
trans-3-decene- 1,1O-4ioic
acid #{149}
trans-3-dodecene1,12-dloic acid #{149}
fatty acid metabolism #{149}
gas chromatography #{149}
mass spectrometry
#{149}
inherited disorders #{149}
impaired /9-oxidation
In a previous report (1), we described a case of a child
who developed a severe metabolic acidosis of unknown
origin and died 26 h after birth. The parents had previously had a child who died in a similar way. The unusual clinical picture and the fact that two children to
consanguineous
parents were afflicted suggested a
metabolic disorder. We then found lactic acid and a
series of aliphatic dicarboxylic acids, six to 14 carbon
atoms in chain length, when the urine of the second
child was analyzed by gas chromatography/mass
spectrometry. This strengthened
the suspicion of a metabolic disorder.
Department
of Clinical Chemistry, University of Gothenburg,
Sahigren’s Hospital, S-413 45 Gothenburg, Sweden.
Received Feb. 13, 1976; accepted May 18, 1976.
1330 CLINICALCHEMISTRY,Vol. 22, No. 8. 1976
Here, we report the synthesis of reference compounds
and the isolation and complete structural analysis of a
series of mono-unsaturated
dicarboxylic
acids possessing either cis-5 or trans-3 double bonds. We also
discuss possible metabolic events that could result in
the excretion of this type of acid and in a metabolic acidosis.
Materials and Methods
Analytical
Methods
Extraction
and esterification
of organic acids. Urine
was acidified to pHi with 6 mol/liter HC1 and extracted
three times with three volumes of diethyl ether. The
extract was dried over anhydrous Na2SO4 (5 g/100 ml),
reduced in volume, and treated with diazomethane
in
ethereal solution. Diazomethane was prepared (2) from
N-nitrosotoluene-4-sulfomethylamide
(Fluka AG,
Buchs, St. Gallen, Switzerland).
Blood serum was acidified to pH 1 with 1 mol/liter
HC1. Twenty volumes of chloroform/methanol
(2/1)
were added and the solution was stirred for 1 h at room
temperature.
After filtration, the filter paper with residue was extracted with 10 volumes of the same solvent
mixture. Six volumes of water were added to the combined extracts, for partition.
The upper phase was
reextracted
once with one volume of chloroform/
methanol/water
(86/14/1, by vol) (3). The combined
lower phases were evaporated
and the residue was
treated with diazomethane.
Silicic acid column chromatography.
Two kinds of
column fillings were used: (a) silicic acid, 100 mesh
(Mallinckrodt
Chemical Works, St. Louis, Mo. 63160)
dried at 120 #{176}C
overnight, and (b) silicic acid mixed with
AgNO3 in the proportions
4/1 (by weight) and then
dried as above (4). The columns were packed in diethyl
ether/petroleum
ether (bp, 40-60 #{176}C),
1/9 by vol, and
the samples were applied in the same solvent. The load
was 1-10 mg per gram of silicic acid, and the elution was
performed by step-wise increase in the proportion
of
diethyl ether to a final ratio of diethyl ether/petroleum
ether of i/i.
Thin-layer
chromatography.
Analytical thin-layer
chromatography
was performed on silica gel G (Merck
AG, Darmstadt, West Germany) containing AgNO3, 100
g/kg (5). The plate (layer thickness, 0.25 mm) was activated at 120 #{176}C
for 30 mm and the solvent used was
a mixture of diethyl ether/petroleum
ether (30/60, by
vol). The chromatogram
was developed for 50 mm; the
plate was sprayed with a solution of 30 g of cupric acetate per liter of phosphoric acid (80 mI/liter) and then
heated at 220 #{176}C
for 30 mm (6).
Infrared
spectrometry.
Infrared spectra were recorded with a Model 257 spectrometer
(Perkin-Elmer
Corp., Norwalk, Conn. 06856). About 200 g of the
sample was dissolved in 100 tl of chloroform,
and
transferred
into a sodium chloride microcuvette.
Gas chromatography.
The instrument,
Model 5750
(Hewlett-Packard
was equipped
stationary
Instruments,
Avondale,
with a flame ionization
phases
were
3% or 5% silicone
Pa.
19311)
detector.
gum
The
rubber,
in acetonitrile
(1/1, by vol) (Pierce Chemical Co.,
Rockford, Ill. 61105) at room temperature
for 10 mm,
for silylation of hydroxyl groups.
Analysis
of polar urinary compounds.
The urinary
content of polar, low-molecular
compounds,
not extracted into diethyl ether, was analyzed by gas chromatography-mass
spectrometry after esterification
with
butanol-HC1 followed by trifluoroacetylation
(9).
Synthesis of Reference Compounds1
1-Chloro-5-iodopentane
(1). 1,5-Dichioropentane(K & K
Laboratories, Plainview, N.Y. 11803) (141 g, 1 mol) and sodium iodide (150 g, 1 mol) were refluxed with stirring in 1.2
liter of acetone for 7 h. The desired product was obtained by
distillation on a spinning band column. The yield was 94 g,
with bp 103 #{176}C
at 1.6 kPa.
1-Chloro-4-pentyne
(2). Acetylene (52 g, 2 mol) was passed
over KOH and concentrated sulfuric acid into 1 liter of liquid
ammonia.
Freshly cut pieces of sodium (50 g, 2.2 mol) were
dissolved at such a rate that a blue color did not appear (10).
1-Chloro-3-bromopropane
(Fluka) (315 g, 2 mol) was added
dropwise at the temperature of boiling ammonia during 2 h.
The mixture was then stirred for an additional 13 h in an
ethanol/solid CO2 bath. A few grains of NH4C1 was then added
and the ammonia allowed to evaporate. Water, 600 ml, was
then added and the pH was brought to 2 with 6 mol/liter HC1.
The mixture was extracted with diethyl ether. The ether phase
was washed with water, Na2CO3 solution
(50 g/liter) and
OV-17, on 100- to 120-mesh Gas Chrom Q, or 10% ethylene glycol succinate on 100- to 120-mesh Gas Chrom
Q (both from Applied Science Laboratories Inc., State
College, Pa. 16801). The carrier gas was helium, at a flow
rate of 25 ml/min.
For the quantitative
estimation of urinary acids, n
eicosane was added as internal standard to the methwater, and finally dried over Na2SO4. Distillation
on a 30-cm
Vigreux column yielded the desired product, bp 115-116 #{176}C,
ylated diethyl ether extract. The sample was then
in a yield between 50 and 120 g.
subjected to isothermal gas chromatography
on a col1,10-Dichloro-4-decyne
(3). Sodium amide was prepared
umn containing ethylene glycol succinate. Dicarboxylic
from 1.5 liter of liquid ammonia and sodium (28 g, 1.2 mol)
acids (C6 to C14) were measured
at 140 #{176}C
and at 180 #{176}C,with catalysis of 0.3 g of Fe (N03)3-6 H20 (11). 1-Chloro-4and lactic acid at 110 #{176}C.
For the estimation of monopentyne
(2; 102.5 g, 1 mol) was added dropwise at -33 #{176}C
during 1 h. 1-Chloro-5-iodopentane
(1) (233.5 g, 1.1 mol) was
carboxylic and dicarboxylic acids in serum, 20 ig of
then added dropwise with vigorous stirring during 2 h at -33
nonanedioic acid and 40 ig of heptadecanoic
acid were
#{176}C.
The reaction flask was placed in an ethanol/solid
CO2
added as internal standards to 2 ml of serum. After exbath, and the mixture was stirred for 12 h. The ammonia was
traction and methylation
as above, the sample was
then allowed to evaporate and 550 ml of water and a few grams
subjected to silicic acid column chromatography,
with
of NH4C1 was slowly added. The mixture was acidified to pH
1 with HC1 and extracted with diethyl ether. The ethereal
the use of 1 g of silicic acid. Mixtures of diethyl ether
layer was washed with Na2CO3 solution (50 g/liter) and with
and petroleum ether were used to elute monocarboxylic
water, and dried over Na2SO4. Gas chromatography
on a
acid esters (10 ml, 5/95, by vol) and dicarboxylic acid
column of 1% OV-17 showed that the product was essentially
esters (10 ml, 20/80, by vol, followed by 10 ml, 30/70, by
pure. The yield was 30-50 g.
1,10-Dicyano-4-decyne
(4). Sodium
cyanide (120 g, 2.4
vol). Gas chromatography
was performed on an ethylene
mol), which had been dried at 110 #{176}C,
was added with vigorous
glycol succinate column by isothermal runs, at 95 #{176}C
for
stirring to 600 ml of dimethylsulfoxide that had been distilled
medium-chain monocarboxylic acid esters, at 140 #{176}C
for
over CaH2 (12). 1,10-Dichloro-4-decyne
(3) (207 g, 1 mol) was
long-chain monocarboxylic
acid esters and short-chain
then added with stirring at such a rate that the temperature
dicarboxylic
acid esters, and at 180 #{176}C
for mediumdid not exceed 165 #{176}C.
After cooling at room temperature,
the
reaction mixture was added to 400 ml of a saturated NaCl
chain dicarboxylic acid esters.
solution. The mixture was extracted with chloroform, and the
Mass spectrometry.
Mass spectra were recorded with
chloroform phase was washed with a saturated NaCl solution,
a Model 9000 combined
gas chromatograph/mass
and dried over Na2SO4. The product was separated from dispectrometer
(LKB Produkter,
Bromma,
Sweden),
methylsulfoxide
by distillation
on a Vigreux column. The yield
using an acceleration voltage of 3.5 kV and an electron
was 66 g; bp 126-140 #{176}C
at 13 Pa.
acid (5). 1,10-Dicyano-4-decyne (4)
energy of 70 eV. The ion source temperature was 270 #{176}C, 5-Dodecyne-1,12-dioic
(4.55 g, 24 mmol) was refluxed for 7 h in 35 ml of ethanol/water
the separator temperature
300 #{176}C.
The column used
(85/15, by vol) containing 5.5 g of KOH. Water, 30 ml, was
contained 3% OV-17 on Gas Chrom Q.
then added, and the mixture acidified to pH 1 with 6 mol/liter
Hydroxylation
at double bonds. Double bonds in
HC1. Needle-shaped
crystals, 3.2 g, were obtained
after coolunsaturated
compounds
were hydroxylated
with osing; mp 88 #{176}C.
mium tetroxide (BDH Chemicals, Poole, England) (7).
Excess osmium tetroxide
was reduced with sulfur
‘Melting points were recorded in glass capillaries in a heating block.
dioxide in methanol (8). The product was treated with
Elementary analyses were performed by Scandinavian Microanalydiazomethane
for re-esterification
of carboxyl groups,
tical Lab., do L#{246}vens
Kemiska Fabrik, Byvej 11, Ballerup, Copenand then with N,O-bis(trimethylsilyl)-trifluoracetamide
hagen, Denmark.
-
CLINICALCHEMISTRY,Vol. 22,No.8,1976 1331
Analysis: C12H1804; caic.: C, 63.70; H, 8.02; found: C, 63.77;
H, 8.11.
cis-5-Dodecene-1,12-dioic
acid (6). 5-Dodecyne-1,12-dioic
acid (5) (1.75 g, 7.7 mmol) was hydrogenated
in 15 ml of dry
methanol
containing
90 mg of palladium on BaSO4, 50 g/kg,
and 90 mg of quinoline (13).
The calculated amount of hydrogen was consumed during
a 3.5-h period. The solution was filtered through Celite
(Johns-Manville Products, Denver, Cob. 80217), ethyl acetate, 100 ml, was added, and the solution was washed with
water and dried over Na2SO4. Crystallization from ethyl acetate/petroleum ether (1/1 by vol) yielded about 1 g; mp 52-58
OC.
Analysis:
H, 8.58.
C12H20O4; calc.: C, 63.14; H,
8.83; found: C, 62.73;
1,8-Dichloro-4-octyne
(7). Sodium amide was prepared
from 1.2 liters of liquid ammonia and sodium (26 g, 1.1 mol),
catalyzed with 0.3 g of Fe (N03)3.6 H20 (11), during 40 mm.
1-Chloro-4-pentyne
(2) (102.5 g, 1 mol) was then added
dropwise at -33 #{176}C
over a 1-h period, with stirring. The reaction flask was placed in an ethanol/solid CO2 bath and the
reaction mixture was stirred for 1 h. 1-Chloro-3-bromopropane
(157.5 g, 1.0 mol) was then added dropwise during 1 h. The
reaction was followed by gas chromatography on a column of
1% OV-17; when this was complete, the ammonia was allowed
to evaporate. Ammoniuni chloride was added to decompose
unreacted sodium amide. Water, 600 ml, and diethyl ether
were added, and the organic phase was washed with 6 mol/liter
HC1, water, Na2CO3 (50 g/liter) and water, and dried over
Na2SO4. The volume was reduced by evaporation, and the
fraction was distilled on a spinning band column. The yield
was 35 g, with bp 116 #{176}C
at 1.3 kPa.
1,8-Dicyano-4-octyne
(8). Sodium cyanide (30 g, 0.6 mol),
which had been dried at 110 #{176}C,
was added with vigorous
stirring to 150 ml of dimethylsulfoxide that had been distilled
over Call2 (12). 1,8-Dichloro-4-octyne (7) (45 g, 0.25 mob) was
added dropwise with stirring, at such a rate that the temperature did not exceed 160 #{176}C.
The solution was stirred for 10
mm after addition was completed, or until the temperature
of the reaction mixture was below 50#{176}C.
After cooling, chloroform, 150 ml, and saturated NaCl solution, 150 ml, were
added. After partition, the water phase was extracted once
more with chloroform, and the combined chloroform phases
were washed twice with saturated NaCl solution and dried
over Na2SO4. The volume was reduced by evaporation, and
the product was distilled on a spinning band column. The
yield was log; bp 137 #{176}C
at 13 Pa.
5-Decyne-1,10-dioic
acid (9). l,8-Dicyano-4-octyne
(8)
(3.88 g, 24 mmol) was refluxed during 6 h in 35 ml of ethanol/water, 85/15 by vol, containing 5.5 g of KOH. Water, 15
ml, was added and the volume was reduced to 25 nil. Another
30 ml of water, and a few drops of 6 mob/liter HC1 were then
added. White crystalline material was obtained after cooling
with asp 109 #{176}C
[reported mp 109.2-110.1 #{176}C
(14)].
Analysis: C10H1404; caic.: C, 60.59; H, 7.12; found: C, 60.59;
H, 7.26
cis-5-Decene-1,10-dioic acid (10). 5-Decyne-1,10-dioic acid
(9) (1.45 g, 7.3 mmol) was hydrogenated in 15 ml of dry
methanol, containing 75 mg of palladium on BaSO4 (50 g/kg)
and 75 mg of quinoline (13). The calculated amount of hydrogen was consumed in 1 h. The solution was filtered through
Celite, 100mb of ethyl acetate was added, and the solution was
washed with 2 mob/liter HC1, and dried over Na2SO4. The
organic phase was evaporated, and the product was crystallized from benzene/n-heptane (1/1 by vol); mp 76-78 #{176}C
[reported mp 77.4-78.6 #{176}C
(14)].
Analysis: C10H1604; cabc.: C, 59.98; H, 8.05; found: C, 60.20;
H, 7.98
Preparation
of trans-5-olefinic
compounds
from
the cor-
cis-5 compounds. A solution of 10 mg of cis-5decenedioic acid (10) dimethyl ester or cis-5-dodecenedioic
acid (6) dimethyl ester and 10 l of diphenyl sulfide in 12 ml
of benzene was irradiated by ultraviolet light for 16 h (15).
After hydrolysis in KOH (0.1 mob/liter) in water for 4 hat 60
#{176}C,
the products were separated from diphenyl sulfide as free
acids on columns of silicic acid, with the use of diethyl ether/
petroleum ether (1/9 to 8/2, by vob) as ebuent. The isomerized
acids were characterized by thin-layer chromatography, infrared spectrometry, and mass spectrometry of dimethyl esters, as well as of derivatives after osmium tetroxide oxidation.
responding
Results
Gas Chromatography of Organic Acids in Urine
20
RETENTION T1
(P11W
Fig. 1. Gas chromatogram of a methylated diethyl ether extract
of urine
Top cfromato-am:
The present case of lactic acldosis. 1. lactic acId, 2. 2hyox,buty,1c
acid, 3. 3hycfroxybutyrlcacid, 4. hexanediolc acid, 5. octanedlolc
acid, 6. decenedlolc acId, 7. decanedlolc acId, 8. n-elcosane, 9. dodecanedlolc
acid + dodecenedloic acId, 10. tetradecenedlolc
acid. The identification of
compounds in peaks 8, 7, 9, and lOis based on results reported In this work.
Bottom chromatogram:
1. 3-methylglutaconic
acid, 2. hexanedloic acid, 3. 3methylhexanediolc acid, 4. 3.4-methylenehexanedioic acid, 5. 2-ketoglutaric
acid, 6. octenedloic acid + octanedloic acid. 7. cItric acid, 8. 4-hydroxyphenylacetic acid, 9. decenedloic acid + decanedloic acid, 10. unidentified,
11. 5-decynedioic acid, 12. n-eicosane,
13. hippaic acid. The chromatograms
were run under identical conditions except that the first 10 mm of the top chromatogram were recorded at 0.05 the sensitivity. The temperatwe was programmed from initIally 70 to 270 #{176}C,
at a rate of 4 #{176}C/mln
1332 CLINICALCHEMISTRY,Vol. 22, No. 8, 1976
Figure 1 shows a gas chromatogram
of the methyl
esters of organic acids in urine from the present case and
in an equal volume of urine from a normal newborn. The
urine from the patient was more dilute, as judged from
the creatinine concentration
(Table 1), and a number
of normally occurring acids were not detected, e.g., 3methylhexanedioic
acid, 3,4-methylenehexanedioic
acid, citric acid, and p-hydroxyphenylacetic
acid (peaks
3, 4, 7, and 8 in the lower part of Figure 1). The chromatogram from the patients’ urine showed a large peak
(peak 1) caused by lactic acid, followed by smaller peaks
of 2-hydroxybutyric
acid (peak 2) and 3-hydroxybutyric
acid (peak 3). The dominating
feature of the chromatogram was a complex of peaks (peaks 4 to 10), which
all appeared to represent aliphatic dicarboxylic acids.
Comparison
with authentic
reference
compounds
identified peak 4 as hexanedioic acid and peak 5 as octanedioic acid. These acids were also present in the
reference urine (peaks 2 and 6).
:0:
CH3-
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II
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Fig. 2. Massspectrum of the methyl ester of n-decanedioic acid
(top spectrum) and of compound I (bottom spectrum)
Fig. 3.Mass spectrum of the methyl ester of n-dodecanedioic
acid (top spectrum) and of compound/I
(bottom spectrum)
No molecular ion (rn/s 230) is visIble. Some of the major Ions are: rn/s 199 =
M-31, rn/s 166 = M-32-32, rn/s 157 M-73, rn/a 138 = M-32-60, and rn/s
125 = M-32-73
No molecular ion (rn/s 258) Is visible. Some of the major ions are: rn/S 227 =
M-31, rn/s 194 M-32-32, rn/s 185 M-73, rn/s 166 M-32-60, and rn/s
153 = M-32-73
Isolation of Aliphatic
6, 9, and 10. For use as model compounds in the following structural analysis we synthesized cia- 5-decenedioic acid and cis-5-dodecenedioic
acid. Mass spectra
of the methyl esters of compounds
III to VII (Figures
4-7) showed a difference from those of saturated dicarboxylic acid esters in that they contained ions at
M-32 and M-74 rather than at M-31 and M-73, a wellestablished difference between saturated and unsaturated fatty acid methyl esters (17). The molecular ions
were small, but clearly visible, and established that the
parent acids contained one double bond and had the
following chain length: C10, compound
III and compound VI; C12, compound
IV and compound
VII; C14,
=
Dicarboxylic
Acids
For the identification
of compounds in peaks 6 to 10
in the chromatogram
of the patients’ urine (Figure 1)
a diethyl ether extract of 9 ml of the patients’ urine was
fractionated
on a column of silicic acid. Acids with 10
to 14 carbon atoms were eluted with diethyl ether/petroleum ether (1/9 to 3/7 by vol), whereas those with 8
to 6 carbon atoms were eluted together with lactic acid
with diethyl ether/petroleum
ether (3/7 to 4/6, by vol).
The combined C14 to C10 fractions were further separated on a column of silicic acid-silver nitrate, with a
fine stepwise gradient of diethyl ether in ‘petroleum
ether. On this column, seven compounds were resolved,
compounds
I to VII.
Determination
of Structure
Saturated
dicarboxylic
acids. The lower parts of
Figures 2 and 3 show the mass spectra of the methyl
esters of compound
I (part of peak 7 in Figure 1) and
compound
II (part of peak 9 in Figure 1). These spectra
are typical of methyl esters of dicarboxylic acids in that
they show symmetrical
loss of fragments from the two
carboxymethyl
ester ends of the molecule (see Figures
2 and 3). The molecular ions were absent (16). The two
compounds
had mass spectra identical with those of
methyl esters of authentic decanedioic acid and dodecanedioic acid (Figures 2 and 3).
Unsaturated
.dicarboxylic
acids. Mass spectra obtained from the separation shown in Figure 1 indicated
the presence of unsaturated dicarboxylic esters in peaks
=
compound
=
=
V.
To establish the position of the double bond, we hydroxylated methyl esters of compounds
III to VII and
the methyl esters of the two synthetic reference compounds with osmium tetroxide and prepared the tnmethylsilyl derivatives of the vic-diols. Spectra of this
type of compounds are dominated
by ions formed by
cleavage between the vicinal tnimethylsilyloxy
groups
and by ions formed by loss of methanol (32 m.u.) and
CH3)3-Si (73 m.u.) from the two fragments. This type
of fragmentation
is shown in Figures 8 to 10 for the derivatives of compounds
III to V and for the corresponding derivatives of the synthetic reference compounds (Figures 8 and 9). The mass spectra established
the 5-position of the double bond in these acids.
The mass spectra of the methyl esters of compounds
IV and VII after hydroxylation and tnimethylsilylation
CLINICALCHEMISTRY,Vol. 22,No.8,1976 1333
1#{176}; :
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311
591
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Mass spectrum of the methyl ester of synthetic cls-5acid (top spectrum) and of compound III (bottom
spectrum)
FIg. 4.
decenedioic
The molecular ion Is found at rn/s 228. Some of the major Ions are: rn/s 196
= 11-32, rn/s 164 =11-32-32,
rn/s 154 14-74, rn/s 13611-32-60, and rn/s
123
11-32-73
indicated that they contained a 3,4-double bond. The
mass spectrum of the derivative of the C10 acid was
however not very satisfactory. Figure 11 shows the mass
spectrum of the derivatives of the C12 acid, compound
VII. The dominating ion appears at m/e 259, indicating
a 3,4-position of the double bond. However, m/e 175,
expected from the other part of the molecule, was only
a small ion. Instead, there was a large ion at m/e 248. It
could not be explained by a simple cleavage between the
two tnimethylsilyboxy
groups. The ion is probably a rearrangement
ion formed by the expected cleavage
combined with migration of a trimethylsilyl group to the
carboxyl group, i.e. 175 + 73 m.u. This kind of fragmentation
has previously been described for the tnmethylsilyl derivatives of aldaric and aldonic acids (18)
and for the tnimethylsilyl derivatives of the methyl esters of 2,3-dihydroxyhexacosanoic
acid (19). The rearrangement occurs when two vicinal hydroxyl groups are
located close to the carbonyl group. The mass spectrum
of the derivative of compound
VI, by analogy with the
spectrum discussed above, indicated that the parent
compound was a 3-decenedioic
acid.
The geometrical configuration of the double bond was
established by infrared spectroscopy and by thin-layer
chromatography
on silicic acid impregnated
with
AgNO3. Compounds
VI and VII showed distinct absorption peaks at 970 cmi, characteristic
of a transconfiguration
(20), whereas this absorption was absent
in compounds
III to V. Synthetic
cis- and trans- 5decenedioic
acids were well separated
by thin-layer
chromatography,
with RF-values 0.24 and 0.33, respectively. Compound
III migrated with the cia isomer.
1334 CLINICALCHEMISTRY,Vol.22,No.8,1976
00
00
00
LJI
100
(20
214
*41
I
‘*2
I
j’
140
.i__I1i
290
190
I
I
200
220
rn/s
Fig. 5. Mass spectrum of the methyl ester of synthetic c/s-S..
acid (tc spectrum) and of compound IV(bof torn
spectrum)
dodecene-dlolc
The molecular Ion Is found at rn/s 256. Some of the major ions are: rn/s 224
= 11-32, rn/s 192 = 11-32-32, rn/s 182=11-74, rn/s 164 11-32-60. and rn/s
150 = 11-32-74
10;
CH3-OtC
IItcH2tt2)2_
i
‘
(CH2)7-C
CHCH-
II-O-CH3
311
59’
73
I
3.I-
In
z
Ui
5z
SO
Ui
40
14
20
0
20
40
40
II1L2’rTITLT
10
00
ItO
40
ISO
.00
200
220
240
210
210
rn/s
FIg. 6. Mass spectrum of the methyl ester of compound V
The molecular ion is found at rn/s 284. Some of the major Ions are: rn/a 252
0011.32, rn/s 221 = 11-31-32. rn/a 2101W11-74, rn/s 192-11-32-60,
and rn/s
178 00 11-32-74
By the same technique the cis-configuration
was established for compounds
IV and V. No reference compounds were available containing a 3,4-double bond, but
the isolated compounds
VI and VII migrated slightly
faster than the transacids of the same chain leigth.
According to Gunstone et al. (21) this is to be expected
for & isomers when compared to & isomers in this
chromatographic
system. Thus, the observed mobility
supports the infrared-evidence
for a trans-configuration
of compounds
VI and VII.
1I
I
I
o
cH-o-1-C+C=cH-ca97-C-o-cH3
TNS
7*8
I
I
II
CH3-O-C-lCH25-C94-f-CH-IC543l-C-O-CK3
II
311
203
203
4”
7J
5
3.5-
In
S.
SO
z
(U
I-
z
U,
90
74
SO
.L. L.
ISO
90
(U
*4
‘I
S.
2
U
20
O
20
00
00
LLJ
00
00
ItO
40
ISO
[I
ISO
r.L
ISO
290
1.
090
20
U
90
224
220
240
290
210
300
rn/s
1,
Fig.7.Mass spectrumofthemethylesterof compound VII
ion Is found at rn/s 256. Some of the major Ions are: rn/s 224
= M-3Z rn/s 196 (not marked) 0014.60, rn/s 192 = 14-32-32, rn/s 182M-74,
rn/s 164M32_60,
and rn/s 1501W 14-32-74
i1LLL.
1
The molecular
m’s
of the hydroxylated and trlmethylsllylated
derivative
ofthemethyl ester of synthetic cLs-5-decenedlolcacid
Fig. 8. Mass spectrum
(top spectrum) and of the same derivative
of compound III
(bottom spectrum)
Dicarboxylic
Acids in Urine and Serum
Table 1 shows the excretion of dicarboxylic acids in
the present case, in a case of fructose-1,6-biphosphatase
deficiency2 with acidosis and a large excretion of lactic
acid, and in normal newbonns The urmnes from the two
cases of lactic acidosis were more dilute, as judged from
creatinine concentration, and it therefore seemed appropriate
to calculate concentrations
in relation to
creatini#{241}e.
The excretion of dicarboxylic acids in the
urine from the other case of lactic acidosis and in normal
urine was of similar magnitude, whereas in the patient
the excretion of hexanedioic acid and octanedioic acid
was about 10-fold greater and the excretion of C12 and
C14 acids at least 100-fold greater.
The dicarboxylic acids found in urine from the patient were also detected in serum (Table 2), but with the
exception of C6 and C8 acids they were not found in the
serum of three normal newborns. The composition of
the monocarboxylic acid fraction was not significantly
different from that in normal newborns.
Polar Low-Molecular-Weight
Compounds in the Urine
The urine from the present case was also analyzed for
polar coitstituents not extracted into diethyl ether. This
analysis was disturbed
by the presence of tnis(hydroxymethyl)aminomethane,
which had been given to
the child for treatment
of the acid/base disturbance.
However, we found no large, abnormal metabolites
in
this way.
No molecular Ion (rn/s 406) Is vIsible. The original double bond posItIon is revealed by the hiteriee fraent
Ions at rn/s 203. Other ions we: rn/s 375 = 14-31,
rn/s 171 = 203-32, and rn/s 73 = trimethylsllyl ions (TMS
trimethylsilyl-)
INS #{149}
INS
C,O3_O__ICN73_SI_l_fN_ICN2I__O_OS3
203
3)4
NI
90,.
Fig. 9. Mass spectrum of the hydroxylated and trlmethylsllylated
derivative of the methyl ester of synthetic cls-5-dodecenedloic
acid (top spectrum) and of the same derivative of compound IV
(bottom spectrum)
The original double bond posItion Is revealed by the Intense fragmeott Ions at rn/s
203 and 231. No molecular Ion (rn/s 434) Is visIble. Other Ions are: rn/s 403
= 14-31, rn/s 199 = 231-32, and rn/s 171 = 203-32
INS
INS
CO_LICN2S_fHfOIC.LO_C
30)
209
Discussion
Urine from healthy
infants and adults normally
of aliphatic dicarboxylic
acids,
e.g., saturated and monoenic acids of chain length C4 to
C9 (22, 23). Dicarboxylic acids of similar chain length
contains
2Diagia
small amounts
based
Johan Waldenstrom,
on enzyme determinations
performed
by Dr.
at this department (unpublished work).
IS,.
10. Massspectrum of the hydroxylated and lrlmethylsllytated
derivative of the methyl ester of compound V
FIg.
No molecular Ion (rn/s 462)18 seen. The intense Ions at rn/s 259 and 203 show
the origInal double bond positIon. Other Ions are: rn/s 227= 25942, rn/s 171
= 203-32, and ,n/e431 = 14-31
CLINICALCHEMISTRY,Vol. 22, No. 8, 1976
1335
Table 1. Lactic Acid and Dicarboxylic Acids Found in the Urine of the Present Case of Lactic Acidosls
in a Case of Fructose-1,6-biphosphatase Deficiency, and In Eight Normal Newborns
Concentration
Compound
mmol/I
Lactic acid
Hexanedioic
Octanedioic acidd
Decanedioic acidd
Dodecanedloicacid
5-cis-Decenedioic
acid
5-cis-Dodecenedioic
acid
5-cis-Tetradecenedioic
acid
3-trans-Decenedioic acid
3-trans-Dodecenedioic
acid
Creatlnine concentratIon,
o Creatlnine
concentratIon,
mot/mol
Normal nswborns
(n = 8, m.an and rang.)
Fructo..-1,6-blpho.phatas.
d&Ici.ncy
b
p#{149}#{149}j a
mmol/l
cr#{149}atlnln.
20.5
0.12
0.09
0.18
0.24
0.10
0.44
0.23
47.4
0.28
0.21
0.42
0.04
0.09
0.14
0.33
0.56
0.23
1.02
0.53
24.0
mollmol
cr.atlnln.
mmol/l
39.3
0.04
0.02
<0.004
<0.00 1
<0.004
<0.00 1
<0.001
0.07
0.03
<0.007
<0.002
<0.007
<0.002
<0.002
<0.004
<0.00 1
<0.007
mol/mol
C
cr.atlnin.
<0.2
<0.05
0.05(0.01-0.13) 0.01(0.002-0.03)
0.11(0.02-0.19) 0.02(0.005-0.04)
0.06(0.01-0.09) 0.01(0.005-0.025)
<0.01
<0.002
<0.05
<0.01
<0.01
<0.002
<0.01
<0.002
<0.05
<0.01
<0.002
<0.01
<0.002
0.43 mmol/liter
0.61 mmol/llter
#{176}Creatinine
concentratIon 5.9(1.9-11)
mmol/liter
U The values for normal newborns Include monounsattrated
I7
Isomers
ISlIObolic
290
HOOC\/S__\dT._,COOh
HOOC...,COOH
JJ
block
ij
,/A%,COOH
IS,.
-
Fig. 11. Mass spectrum of the hydroxylated and trimethylsilylated
derivative of the methyl ester of compound VII
No molecular Ion (rn/s 434) is visible. The large Ion at rn/s 259 Is derIved from
the above formula by simple cleavage, whereas the Ion at rn/s 248 isa rearrangement ion, 175 + 73 (see text), and rn/s 403 = M-31
Table 2. Dicarboxylic Acids in Serum from the
Present Case and from Normal Newborns
Prssnt
case
Compound
Concn,
Hexanedloic acid
Octanedioic acid
Decanediolc acid
Dodecanediolc acid
c!s-5-Decenedioic acid
cis-5-Dodecenedioic
acid
Normal n.wborns
(nsean, n
3)
imo1/iit.r
2
-‘1
16
30
<2
28
<2
9
<2
cls-5-TetradecenedioIc
acid
24
<2
<1
trans-3-Oecenedioic acid
trans-3-Dodecenedioic
acid
6
17
<2
<2
1336
CLINICAL
61
CHEMISTRY,Vol.22,No.8,1976
oxidation
Fig. 12.Suggestedpathway for formation of dicarboxylic acids
from oleicacidinthe patient studied
containing methyl branches (24), cyclopropanerings
(25), and acetylenic bonds (26) have also been found.
In certain pathological
and experimental
conditions
increased excretion of normal or “unusual” dicarboxylic
acids has been reported. Thus, in ketoacidosis (27) excretion of hexanedioic
and octanedioic
acids is increased (up to 0.5 mol/mol creatinine) and in glycogen
storage disease of types I and III (28) odd- as well as
even-numbered
saturated acids of chain length C6 to C10
are excreted in moderate amounts (0.02 mol/mol creatinine). In a case of carnitine deficiency, Karpati et al.
(29) reported the excretion of large amounts of saturated and monoenic C6 to C8 acids (-4 mol/mol creatinine). Administration
of hypoglycin A (2-methylenecyclopropanealanine)results
in the excretion of unsaturated dicarboxylic acids (30)-cis-4-octanedioic
acid,
cis- 4-decenedioic
acid, and cis -4,7-decadienedioic
acid-apparently
not originating from oleic acid.
The acids mentioned
above presumably
originate
from fatty acids of greater chain lengths. An w-oxidation
occurs when for some reason the fl-oxidation does not
proceed in the usual way. The final metabolite
thus
originates by way of w-oxidation and one or several 3oxidation steps from either end of the carbon chain.
Different causes may start this sequence of events: (a)
steric hindrance in the molecule such as branches or
rings, (b) transport defects such as a decreased inflow
into the mitochondria
in carnitine deficiency, (c) enzyme inhibition as in hypoglycin A treatment, where it
has been shown that methylenecyclopropyl-acetylCoA is formed
and acts as a potent inhibitor of butyryl-CoA dehydrogenase
(31), and (d) a deficiency of an
enzyme in the fl-oxidation cycle.
A decreased activity of one or several enzymes in the
fl-oxidation cycle could result in the formation of dicarboxylic acids of the type found in our patient. We
found no evidence for the presence of trans-2 double
bonds, hydroxyls, or ketones at C-3, and therefore assume a decreased activity of an acyl-CoA dehydrogenase. In pig liver there exist three acyl-CoA dehydrogenases with different chain-length
specificity, one of
which is most active with medium chain acids as substrate (32). Whether a similar situation exists in the
human is not known. The presence of saturated as well
as mono-unsaturated
aliphatic dicarboxylic acids in the
urine reflects the flux of both kinds of fatty acids
through fl-oxidation. The cis- 5-monoenes can easily be
formed from oleic acid as shown in Figure 12. The absence of cis-3-monoenes
may be attributed to the existence of an enzyme that isomerizes this kind of acids
into trans-2-compounds
(33), which are able to circumvent the metabolic block. We are unable, at the
present,
to explain the formation
of the trans-3monounsaturated
compounds. Possibly, another kind
of isomerase has been acting on the cis-3-compounds,
as such enzyme activities have been described (34).
Of the possible causes for an impaired i3-oxidation
mentioned above, the presence of a steric hindrance can
be excluded. Unfortunately,
no carnitine determinations were performed, but the clinical picture was different and the urinary acids were of longer chain length
than those described in the carnitine deficiency syndrome. The possibility exists that the lactic acidosis
affected fl-oxidation. Apparently,
however, lactic acidosis, for instance caused by glycogen storage disease
(28), fructose-1,6-biphosphatase
(EC 3.1.3.11) deficiency (Table 1), or of unknown origin (35) is not accompanied by excretion of the type of acids observed
in our case.
Why is lactic acid formed if there is a primary defect
of the fl-oxidation?
One possibility is an intramitochondrial trapping of CoA by accumulating
acyl-CoA,
which would influence the oxidative decarboxylation
of pyruvate. Alternatively, the dicarboxylic acids formed
may be inhibitors, e.g., of the enzymes involved in gluconeogenesis. 4-Pentenoic acid is a potent inhibitor of
liver pyruvate metabolism,
but it is not yet obvious
whether this is due to CoA-trapping
or to enzyme inhibition (36).
In summary,
clinical evidence and biochemical
findings make it tempting to suggest that we are dealing
with a genetic defect with impaired ability for fl-oxidation of fatty acids, presumably located at the acyl-CoA
dehydrogenase step in the degradation of medium-chain
fatty acids.
This work was supported
Medical
Research
by Grant
13X-585 from the Swedish
Council.
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