Analysis of the Citric Acid Cycle Intermediates Using Gas Chromatography-Mass Spectrometry

Article (PDF Available)inMethods in molecular biology (Clifton, N.J.) 708:147-57 · January 2011with183 Reads
DOI: 10.1007/978-1-61737-985-7_8 · Source: PubMed
Abstract
Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.

Figures

Chapter 8
Analysis of the Citric Acid Cycle Intermediates Using Gas
Chromatography-Mass Spectrometry
Rajan S. Kombu, Henri Brunengraber, and Michelle A. Puchowicz
Abstract
Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to
identifying unexpected correlations between apparently related and unrelated pathways of metabolism.
Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer
useful information to understanding interrelationships between the CAC and metabolic pathways under
various physiological and pathological conditions. This chapter presents a relatively simple method that is
sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and
other related intermediates of energy metabolism using gas chromatography-mass spectrometry.
Key words: Citric acid cycle, CAC intermediates, GC-MS, metabolomics, mass spectrometry.
1. Introduction
In mammalian cells, the citric acid cycle (CAC) is a series of
enzyme-catalyzed chemical r e actions that result in the generation
of reducing equivalents (NADH/NAD
+
,FADH
2
/FADH) that
fuel oxidative phosphorylation (mitochondrial cellular respira-
tion), as well as carbon dioxide (1). The CAC links key metabolic
pathways, such as glycolysis (2), gluconeogenesis (3, 4), and
anaplerosis/cataplerosis (57), and provides precursors for many
compounds including fatty acids and some amino acids.
This chapter presents a qualitative method that is sensitive
for simultaneously measuring concentrations of CAC interme-
diates (relative and absolute) and other related intermediates of
energy metabolism using gas chromatography-mass spectrome-
try (GC-MS) and stable isotope technologies. This approach has
T.O. Metz (ed.), Metabolic Profiling, Methods in Molecular Biology 708,
DOI 10.1007/978-1-61737-985-7_8, © Springer Science+Business Media, LLC 2011
147
148 Kombu, Brunengraber, and Puchowicz
been utilized for over two decades in laboratories conducting
classical metabolomics, often in parallel with other biochemi-
cal analyses that use GC-MS techniques (2, 8, 9). With major
advancements in GC-MS technology (increased sensitivity and
throughput), database information from the National Institute
of Standards and Technology (NIST), and cost-effectiveness,
this approach has become more popular to researchers and
thus prompted a new generation of scientists interested in
metabolomics as a tool for the identification of unexpected cor-
relations between apparently related and unrelated pathways of
metabolism (10, 11). We describe a quantification method for
assaying concentration profiles of (water-soluble) CAC metabo-
lites as measured against a mixture of stable isotope reference
compounds, such as [U-
13
C
6
]citrate, [U-
13
C
4
succinate], and
3-hydroxy[
2
H
4
]glutarate (12).
Briefly, our approach utilizes the rapid reaction of sily-
lating reagent with alcohols, acids, and amines to form
silyl derivatives. Commercially, silylating reagents are avail-
able as combinations to accelerate the reaction, as well as
to react with the hindered group. We use either a mix-
ture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and
trimethylchlorosilane (TMCS) to form a TMS derivative or a
mixture of N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide
(MTBSTFA) and t-butyldimethylchlorosilane (TBDMS) to form
a TBDMS derivative (see Note 1). The resultant TMS and
TBDMS derivatives of these metabolites/intermediates can be
performed on plasma, tissue extracts, organ perfusates, or cell
media pr eparations. As previously described (slightly modified
method) (12), the assay involves (i) spiking the sample with one
or more internal standar ds, (ii) homogenization with Folch’s sol-
vent or an acid–methanol mixture and then centrifugation, (iii)
evaporation of extract and derivatization with either trimethyl-
or t-butyldi-silyl, and (iv) identification of the m/z of each
analyte using ammonia-positive chemical ionization (CI) or elec-
tron impact ionization (EI) GC-MS. To analyze compounds con-
taining “keto groups,” such as α-ketoglutarate and oxaloacetate,
we also describe derivatization of TMS-methoxylamine, which
requires a few additional preparatory steps. This chapter does
not present mass isotopomer distribution analysis (MIDA) or
the use of labeling patterns of intermediates to determine fluxes
or turnover, as this approach often requires the use of low iso-
topomer background derivatizing agents (3, 4).
2. Materials
2.1. Chemicals and
Materials
1. Chloroform.
2. Methanol.
Analysis of the Citric Acid Cycle Intermediates 149
3. Glacial acetic acid (99.7%).
4. Selected internal standards: [
13
C
6
]citric and [
13
C
4
]succinic
acids (98%) (Isotec, Miamisburg, OH); (RS)-3-hydroxy-
[
2
H
4
]glutarate was prepared as previously described (13).
5. Regisil
R
+ 10% TMCS (TMS; Regis Technologies, Morton
Grove, IL). Store in a refrigerator (4
C) in a desiccator.
6. N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide
(MTBSTFA +t-BDMCS; TBDMS; Regis Technologies,
Morton Grove, IL). Store in a refrigerator (4
C) in a
desiccator.
7. 10 N sodium hydroxide solution. Store at room tempera-
ture.
8. Methoxylamine–HCl solution (MOX; 100 mM): To pre-
pare MOX solution, weigh 83.52 mg of methoxylamine
hydrochloride in a conical tube and add 10 mL of water.
Adjust the pH to 8–10 using 10 N sodium hydroxide solu-
tion (2). Prepare MOX solution fresh on the day of analysis
(Caution: corrosive; avoid contact with skin and eyes; avoid
breathing; and wear protective clothing) (see Note 2).
9. Chloroform/methanol mixture (2:1, v/v). This mixture is
stable at room temperature and can be used for 1 month.
This mixture is used in extraction method A, which is
slightly modified from Yang et al. (12).
10. Methanol/water mixture (3:2, v/v). This mixture is stable
at room temperature and can be used for 1 month.
11. Acid–methanol mixture: 5% acetic acid in methanol/water
(1:1, v/v). This solution is stable at room temperature and
can be used for 1 month. This mixture is used in extraction
method B (14, 15).
12. Heating block capable of heating up to 70
C.
13. Turbovap evaporator (Caliper Life Sciences, Hopkinton,
MA).
14. Speedvac vacuum concentrator (Thermo Scientific,
Bellefonte, PA).
15. Omni General Laboratory Homogenizer (Kennesaw, GA).
16. Disposable centrifuge tube, polypropylene (15 mL; Fisher
Scientific, Fair Lawn, NJ).
17. Disposable culture tube, borosilicate glass (13 × 100 mm;
Fisher Scientific).
18. Wide opening glass crimp vial (2 mL; Agilent Technolo-
gies, Santa Clara, CA).
19. Glass vial insert (250 μL; Agilent Technologies).
20. Aluminum crimp cap with PTFE/rubber septa (11 mm;
Agilent Technologies).
150 Kombu, Brunengraber, and Puchowicz
2.2. GC-MS
1. Mass spectrometer: Agilent 5973 mass spectrometer, linked
to a model 6890 gas chromatograph equipped with an
autosampler (Agilent Technologies, Santa Clara, CA).
2. Carrier gas: helium (1 mL/min) with a pulse pressure of 40
p.s.i.g.
3. Column: VF-5MS EZ guard capillary column (60 m ×
0.25 mm inner diameter) with guard column (10 m) (Varian
Technologies, Palo Alto, CA).
4. Chemical ionization gas (CI mode): Ammonia
3. Methods
3.1. Tissue
Preparation
The tissues are extracted by two methods, either using chlo-
roform/methanol (method A) (12) or using 5% acetic acid in
methanol/water (method B) (14, 15). Method A is advanta-
geous for simultaneously analyzing CAC intermediates, as well as
cholesterol and fatty acids fr om the same tissue as the chloroform
phase contains these lipids. Method B is specifically useful for
CAC intermediates as it completely extracts these water-soluble
acids from the tissue. Both methods can be used on tissues such
as liver, brain, muscle, and kidney.
3.1.1. Method A:
Chloroform/Methanol
Extraction Procedure
1. Weigh about 0.2–0.5 g of powdered frozen (–80
C) tissue
in a pre-weighed/tarred, 15 mL disposable centrifuge tube
previously chilled over dry ice and record the tissue weight.
(Caution: dry ice is extremely cold and can cause frost bite.
Wear protective gloves and goggles while handling.)
2. After weighing, spike the powdered frozen tissue with the
selected internal standards (~50 nM of [
13
C
6
]citrate, ~30
nM of [
13
C
4
]succinate, and ~30 nM of (RS)-3-hydroxy-
[
2
H
4
]glutarate; see Note 3).
3. Using an Omni homogenizer, homogenize the tissue with
6 mL of pre-cooled (–25
C) chloroform/methanol (2:1,
v/v) for 5 min on a dry ice–acetone bath (caution: dry ice
is extremely cold and can cause frost bite. Wear protective
gloves and goggles while handling) (see Note 4).
4. To break the phases, add 2 mL of ice-cold water to the
same tube and re-homogenize for 5 min at –25
C.
5. Centrifuge the slurry at 670×g for 20 min at 4
C.
6. Collect the upper methanol/water phase in a disposable
culture tube; if necessary, filter to remove tissue fragments.
7. For complete extraction of analytes, a second extraction
is suggested: to the lower organic chloroform phase, add
Analysis of the Citric Acid Cycle Intermediates 151
3 mL of pre-cooled (–20
C) methanol/water (3:2, v/v)
and vortex for 5 min.
8. Repeat steps 5 and 6 (see Note 5).
9. Combine the two upper methanol/water phase extracts
obtained from steps 6 and 8.
10. To the combined extract, add methoxylamine–HCl solu-
tion to a final concentration of 200 μM(see Note 6).
11. Adjust the pH to 8.0 with 10 N sodium hydroxide to com-
plete the derivatization process.
12. Evaporate the extract completely under vacuum overnight
using a Savant vacuum centrifuge (see Note 7).
13. Add 100 μL of TMS (or TBDMS) to the completely dried
residue using a transfer pipette (caution: TMS and TBDMS
derivatizing agents are volatile, toxic, carcinogenic, and
corrosive. Use protective measures like gloves and hood)
(see Notes 8 and 9).
14. Heat the mixture at 70
C for 50 min on a heating block;
this process yields TMS (or TBDMS) and methoxam-
ates/TMS (or TBDMS) derivatives of the analytes (see
Note 10).
15. Allow the sample to cool to room temperature (caution:
avoid moisture).
16. Transfer the derivatized mixture to a wide open glass crimp
vial with glass vial insert and cap tightly.
17. Inject the mixture on to the GC-MS for analysis (see Note
11; see Section 3.3).
18. For quantification of analytes, prepare stock solutions with
known amounts of standards in water. Prepare a serial dilu-
tion. Treat the standards the same way the samples are
treated. The amounts of selected internal standards need
to be the same as that added to the samples. This enables
the amounts of analytes in the sample to be quantified as
absolute or relative amounts. For the derivatization proce-
dure, follow Section 3.1.2, steps 10–17.
19. Plot a standard curve of the peak areas (standards against
internal standards, relative to known amounts of the added
standards) (12).
20. Calculate the absolute or r elative concentrations of analytes
of interest as identified during electron ionization runs (see
Section 4).
3.1.2. Method B:
Acidified
Methanol/Water
Extraction Procedure
1. Weigh about 0.2–0.5 g of powdered frozen (–80
C) tissue
in a p re-weighed/tarred, 15 mL conical “homogenizing”
tube previously chilled over dry ice and record the tissue
152 Kombu, Brunengraber, and Puchowicz
weight. (Caution: dry ice is extr emely cold and can cause
frost bite. Wear protective gloves and goggles while han-
dling.)
2. After weighing, spike the powdered frozen tissue with the
selected internal standards (~50 nM of [
13
C
6
]citrate, ~30
nM of [
13
C
4
]succinate, and ~30 nM of (RS)-3-hydroxy-
[
2
H
4
]glutarate; see Note 3).
3. Using an Omni homogenizer, homogenize the tissue with 5
mL of 5% acetic acid in methanol/water (1:1, v/v) extrac-
tion buffer (chilled on ice) for 2 min on ice bath.
4. Centrifuge the homogenate at 670×g for 30 min at 4
C.
Decant the super natant into a glass test tube and save on ice
and process immediately or f reeze the supernatant at –80
C
until derivatization procedure (see Notes 12 and 13).
5. For the derivatizing procedure, pipette 100–200 μLofthe
supernatant collected in step 3 or use all of the supernatant
collected in step 3 and follow Section 3.1.1, steps 10–20.
3.2. Plasma and
Organ Perfusate
Preparation
1. Thaw the plasma or perfusate on ice at 4
C.
2. Once thawed, immediately aliquot 0.1 mL of the perfusate
or plasma and spike with the selected internal standards (~50
nM of [
13
C
6
]citrate, ~30 nM of [
13
C
4
]succinate, and ~30
nM of (RS)-3-hydroxy-[
2
H
4
]glutarate, see Note 3). The
reserve can be stored in a deep freezer at –80
C for further
analysis.
3. To derivatize analytes containing keto groups, add
methoxylamine–HCL solution to a final concentration of
200 μM, cap tightly, and heat the mixture at 60
Cfor3h.
4. Then add 1 mL of cold acetonitrile/methanol (7:3, v/v) and
vortex for 30 s.
5. Centrifuge at 670×g for 20 min at 4
C.
6. After centrifugation, collect the supernatant and follow the
procedure in Section 3.1.1, steps 12–20.
3.3. GC-MS Assays
1. Following the sample preparation procedur es above (see
Section 3.1.1, step 16), the derivatized samples can be ana-
lyzed either in CI mode (12) or in EI mode (2). The param-
eters described here are for CI mode and the parameters for
EI mode are given in parenthesis.
2. On the Agilent 5973 mass spectrometer linked to a model
6890 gas chromatograph, set the injector temperature at
270
C (EI: 250
C) and the transfer line at 280
C(EI:
300
C).
3. Set the ion source and the quadrupole at 150
C.
Analysis of the Citric Acid Cycle Intermediates 153
Table 8.1
Chemical and electron ionization mass spectra of the TMS or TBDMS derivatives
of CAC and related intermediates
PCI-MOX-TMS
a
EI-TMS
b
EI-TBDMS
b
Analyte Retention time
m/z
to monitor
m/z
to monitor
m/z
to monitor
Succinate 8.55 280 247 331, 289, 215
Fumarate 9.04 278 245 329, 287, 245
Oxaloacetate 10.46 323 290
Malate 10.72 368 335, 245, 233 419, 403, 287
α-ketoglutarate 11.86 337 304, 275 431, 446
Citrate 14.48 481 465, 375, 347 459, 431, 357
Glutamate 348, 246, 230 432, 330, 272
Glutamine 347, 245, 229 431, 329, 271
For the internal standards, the m/z of [
13
C
6
]citrate yields M+6 citrate, [
13
C
4
]succinic acid yields M+4 succinate, and
3-hydroxy-[
2
H
4
]glutarate yields 437, 304, and 260 as TBDMS derivatives.
a
Retention times correspond to the CI method as described in Section 3.1.1 (12).
b
For EI methods, the retention times are relative according to the GC gradient program (this method uses
hydroxylamine instead of MOX) (2)andthem/z of the major fragments for each analyte are given and confirmed by
NIST. The m/z ions are selected based on the spectral characterization (fragmentation pattern) of the particular
derivative as well as their intensities. The intensites of these ions may differ depending on the mass spectrometer used.
4. The GC temperature program is as follows: start at 80
C,
hold for 1 min, increase by 10
C/minto320
C, hold at
320
C for 5 min. (EI: start at 80
C, increase by 5
C/min
to 250
C followed by an increase by 50
C/minto300
C,
hold at 300
Cfor5min).
5. Adjust the ammonia pressure to optimize peak areas.
6. The retention times and m/z values monitored using the CI
method are listed in Table 8.1 and Fig. 8.1.
3.4. Calculations
1. The raw mass spectrometric areas are used to calculate
the analytical parameter (area of analyte)/(area of reference
compound). This parameter is not a relative concentration;
thus, to calculate relative concentrations, use the following
equation:
Relative concentration = average[(area of analyte)/
(area of reference compound)]
i
/average [(area of analyte)/
(area of reference compound)]
c
where i is the intervention/experimental group and c is the
control group.
154 Kombu, Brunengraber, and Puchowicz
Fig. 8.1. Positive chemical ionization chromatograms of metabolites extracted from a liver perfused with 5 mM lac-
tate (adapted from Yang et al. (12)). In addition to
m/z
spectra, the retention times and relative peak heights are also
used to characterize and reference each metabolite. For example, apart from the analytes listed in Table 8.1, the TMS
derivatives of related metabolites such as glycine, aspartate, glucose, pyruvate, phosphoenolpyruvate, dihydroxyacetone
phosphate, glyceraldehyde-3-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, fructose-6-phosphate, and glucose-
6-phosphate can be simultaneously analyzed by introducing appropriate ions and retention times in the scan table of the
mass spectrometer software (panel A: 1 μL split 10:1; panel B: 2 μL splitless,
see
Note 13).
2. Absolute concentrations are calculated against a standard
curve that contains the same amount of internal standard(s)
as added to the samples. Preparation of standard curve: pre-
pare graded amounts of known standards and add a con-
stant amount of internal standard to each of the prepared
standards (reference compound). Run the standards on the
GC-MS along with the samples under the same conditions.
Calculate the ratio of the peak height or area for standards
and samples to that of the internal standard. Using the stan-
dard curve, the concentrations per sample can be calculated
Analysis of the Citric Acid Cycle Intermediates 155
(9). The mass spectrometer software has the function to
build the calibration curves and calculate the concentrations
based on the input to the software.
4. Notes
1. The advantage of TBDMS derivatization is that these
derivatives are more stable to moisture and reduce the risk
of enol silyl ether formation (9, 16). The advantage of the
trifluroacetamide groups in these reagents is their volatil-
ity, low reactivity, and specificity to react only with alcohols
when there is a carboxyl group in the same molecule (16).
2. Instead of MOX, hydroxylamine can be substituted.
3. The estimated amount of internal standards used depends
on tissue type. For example, α-ketoglutarate content is
greater in liver than in brain. For accuracy, it is very impor-
tant to add internal standards at the initial processing
step, otherwise volume additions or losses would need to
be accounted for and the final concentration calculations
adjusted.
4. During the 5-min extraction, the tube is partially immersed
in acetone kept at –25
C by periodic addition of dry ice.
5. The chloroform phase can be used for analyzing fatty acids
and cholesterol (12).
6. This step is to protect/stabilize the keto gr oup by deriva-
tizing with MOX. Steps 10 and 11 can be omitted if the
analytes of interest do not contain keto groups, as this step
is essential for stabilizing the keto group.
7. To shorten the processing time, heat at 50
Cfor3hand
then dry under nitrogen at 50
C using a Turbovap. Take
care not to overheat as it will lead to degradation of ana-
lytes. The reagents in Section 3.1.1, step 13 are highly
moisture sensitive and thus the sample should be com-
pletely dry before proceeding.
8. Make a small hole in the cap with a 22 gauge needle to
avoid pressure buildup and popping of the cap.
9. TMS and TBDMS derivatizing reagents react readily with
glass and plastics. While drawing the reagent using the
syringe, it may block it. It is better to use a transfer pipette.
If a glass syringe is used, wash immediately with methanol
and designate the syringe for these reagents.
10. If moisture or alcohol (protic solvent) is present, the
reagents will first r e act with them to form a white residue.
156 Kombu, Brunengraber, and Puchowicz
Avoid opening the reagent bottle frequently and, once
opened, do not use it after a month or two. These reagents
are volatile; avoid over heating to prevent complete dry-
ness. Switch on the heating block at least an hour earlier
before placing the tubes to get the block equilibrated at
70
C.
11. The injection volume and split flow may need to be
adjusted according to the intensity of the individual analyte
of interest. For example, pyruvate is less intense compared
to malate, thus requiring more injection volume without a
split flow.
12. To ensure the glass test tube does not break upon freezing,
make sure that the volume in the test tube is less that 50%
of the height and store on a slight angle in –80
C freezer.
13. In some procedures, the first fraction is used for other
assays such as for acyl-CoA profiles (14, 15).
Acknowledgments
This work was supported, in whole or in part, by National
Institutes of Health Roadmap Grant R33DK070291 and Grant
R01ES013925. This work was also supported by a grant from
the Cleveland Mt. Sinai Health Care Foundation. We acknowl-
edge the Mouse Metabolic Phenotyping Center (MMPC) at Case
Western Reserve University where many of these procedures were
developed.
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133–153.
    • "GABA and glutamate (Glu) were measured in brain homogenate at Case Western Reserve University, Mouse Metabolic Phenotyping Center. This approach enabled metabolites to be measured with a high degree of sensitivity (Yang et al., 2008; Kombu et al., 2011; Zhang et al., 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: Angelman syndrome (AS) is a rare genetic and neurological disorder presenting with seizures, developmental delay, ataxia, and lack of speech. Previous studies have indicated that oxidative stress-dependent metabolic dysfunction may underlie the phenotypic deficits reported in the AS mouse model. While the ketogenic diet (KD) has been used to protect against oxidative stress and has successfully treated refractory epilepsy in AS case studies, issues arise due to its strict adherence requirements, in addition to selective eating habits and weight issues reported in patients with AS. We hypothesized that ketone ester supplementation would mimic the KD as an anticonvulsant and improve the behavioral and synaptic plasticity deficits in vivo. AS mice were supplemented R,S-1,3-butanediol acetoacetate diester (KE) ad libitum for eight weeks. KE administration improved motor coordination, learning and memory, and synaptic plasticity in AS mice. The KE was also anticonvulsant and altered brain amino acid metabolism in AS treated animals. Our findings suggest that KE supplementation produces sustained ketosis and ameliorates many phenotypes in the AS mouse model, and should be investigated further for future clinical use.
    Full-text · Article · Aug 2016
    • "After 13 C pulse incubations, mitochondrial suspensions (mitochondria in assay medium) were immediately quenched with cold methanol, to minimize metabolite loss during further manipulations. Following extraction, metabolites were derivatized using an optimized procedure to limit potential degradation [31]. First, alpha-ketoacids were methoximated to stabilize known labile species, such as pyruvate, alpha-ketoglutarate and oxaloacetate [14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondria are a focal point in metabolism, given that they play fundamental roles in catabolic, as well as anabolic reactions. Alterations in mitochondrial functions are often studied in whole cells, and metabolomics experiments using 13C-labeled substrates, coupled with mass isotopomer distribution analyses, represent a powerful approach to study global changes in cellular metabolic activities. However, little is known regarding the assessment of metabolic activities in isolated mitochondria using this technology. Studies on isolated mitochondria permit the evaluation of whether changes in cellular metabolic activities are due to modifications in the intrinsic properties of the mitochondria. Here, we present a streamlined approach to accurately determine 13C, as well as 12C enrichments in isolated mitochondria from mammalian tissues or cultured cells by GC/MS. We demonstrate the relevance of this experimental approach by assessing the effects of drugs perturbing mitochondrial functions on the mass isotopomer enrichment of metabolic intermediates. Furthermore, we investigate 13C and 12C enrichments in mitochondria isolated from cancer cells given the emerging role of metabolic alterations in supporting tumor growth. This original method will provide a very sensitive tool to perform metabolomics studies on isolated mitochondria.
    Full-text · Article · Apr 2014
    • "In recent years, several techniques have emerged that could measure CAC intermediates with greater precision, albeit still with limited coverage. In particular these include LC–MS/MS techniques developed by Luo et al. 2007 (Luo et al. 2007) and Koubaa et al. 2013 (Koubaa et al. 2013), GC/MS methodologies (Dunn and Winder 2011; Kombu et al. 2011), HPLC-fluorescence methodology (Kubota et al. 2005), capillary electrophoresis coupled to a mass spectrometer (CE/MS) (Soga et al. 2003; Wakayama et al. 2010), as well as 1 H NMR (Xu et al. 2011). 1 H NMR permits a greater coverage of the CAC intermediates as well as other metabolites (Xu et al. 2011) and does not require isotopically labeled internal standards. However, it is limited by the low micromolar sensitivity and potential spectral overlap. "
    [Show abstract] [Hide abstract] ABSTRACT: The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates. Electronic supplementary material The online version of this article (doi:10.1007/s11306-013-0521-1) contains supplementary material, which is available to authorized users.
    Article · Oct 2013
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