FATTY ACYL CoA
Because the acyl-CoA are present in
nanomole amounts in tissues, and the common glycerol esters are present in
micromole amounts, a very selective assay procedure is required to determine
specifically the whole content of these thioesters or the types of acyl chains
in the CoA esters.
To allow optimum recoveries, adapted tissue extraction procedures must be used,
frequently followed by a concentration or purification step before any
chromatographic analysis.
EXTRACTION
Samples may be quenched rapidly in cooled aqueous solutions of perchloric (Corkey
BE et al., Anal Biochem 1981, 118, 30) or sulfosalicylic acid (Demoz A et
al. J Chromatogr 1993, 635, 251).
Most frequently, tissue samples are extracted in organic solvents using
techniques derived from that of Bligh-Dyer where acyl-CoA are harvested in a
methanolic aqueous phase while the less polar lipids remain in the
chloroform-rich phase (Prasad MR et al., Anal Biochem 1987, 162, 202; Mangino
MJ et al., J Chromatogr 1992, 577, 157; Rosendal J et al., Anal Biochem 1992,
207, 63).
Alternative extraction procedures, derived from that described by Mancha M et
al. (Anal Biochem 1975, 68, 600) are based on the use of buffered
2-propanol followed by washings to remove lipid contaminants and solvent
partitioning to purify acyl-CoA (Woldegiorgis G et al., Anal Biochem 1985,
150, 8; Larson TR et al., The plant J 2001, 25, 115;
Golovko MY et al., J Lipid
Res 2004, 45, 1777).
A better recovery of acyl-CoA was obtained in adding an acyl-CoA-binding
protein to the last aqueous buffered solution (Rosendal J et al., Anal
Biochem 1992, 207, 63).
We give below the main steps of the typical extraction procedure according to
Golovko MY et al. (J Lipid Res 2004, 45, 1777) :
Frozen powdered tissue was homogenized in 2
ml of 100 mM KH2PO4 containing 16 nmol of
heptadecanoyl-CoA as an internal standard. Then, 2.0 ml of 2-propanol was added,
the sample was homogenized again in a glass homogenizer, and 0.25 ml of
saturated NH4SO4 and 4.0 ml of acetonitrile were added.
This mixture was vortexed for 5 min. This solution was subjected to 5 min of
centrifugation (1,900 g ), and the upper phase containing the acyl-CoA was
removed and diluted with 10 ml of 100 mM KH2PO4 (pH 4.9).
All operations were done quickly over a 10-12 min period before centrifugation
in ice-cold conditions. Acyl-CoA were extracted a second time from the tissue
residue according to the same procedure.
As the recovery depends largely on the chain length, a general procedure for the isolation of a wide range of acyl-CoA esters has been reported (Minkler PE et al., Anal Biochem 2008, 376, 275). The described procedure is based on a tissue extraction using acetonitrile/2-propanol mixture and a purification using a SPE column packed with an adapted anion-exchange bed.
PURIFICATION
In several reports the analytical procedure includes a solid-phase purification
step using either an oligonucleotide purification cartridge from Applied
Biosystems (Deutsch J et al., Anal Biochem
1994, 220, 321; Golovko MY et al., J
Lipid Res 2004, 45, 1777), an alumina column (Woldegiorgis G et al., Anal Biochem 1985, 150, 8; Prasad
MR et al., Anal Biochem 1987, 162, 202), or a C18 cartridge (SepPak) (Molaparast-Saless
F et al., Lipids 1988, 23, 490; Mangino MJ et al., J Chromatogr 1992, 577, 157).
ANALYSIS
Gas chromatographic methods were rarely used to analyze fatty acyl-CoA in
biological samples. An application for liver tissue was reported by Prasad MR et
al. (Anal Biochem 1987, 162, 202). The CoA esters are reduced with
sodium borohydride to the corresponding alcohols that are derivatized before
analysis. No more than 50 mg of dry powdered liver was sufficient to obtain
reliable results.
The most frequently used separation method is reversed-phase HPLC with gradient
elution (buffered methanol or acetonitrile) in combination with UV detection.
A highly sensitive and selective method was described for the quantitative
estimation of acyl-CoA (C4 to C20) from plant tissues (Larson TR et
al., The plant J 2001, 25, 115). The method is said to detect acyl CoA
esters down to concentrations as low as 6 fmol in extracts. Acyl CoA are
derived to their fluorescent acyl etheno-CoA in the presence of
chloroacetaldehyde, separated by ion-paired reversed-phase HPLC, and detected fluorometrically.
We give below details on a typical HPLC separation procedure according
to Golovko MY et al. (J Lipid Res 2004, 45, 1777) :
HPLC of acyl-CoA molecular species was
carried out using a C-18 column. The eluent was monitored at 260 nm. The solvent
program for elution was established to resolve the most common polyunsaturated
acyl-CoAs (22:6-CoA, 20:4-CoA, and 18:2-CoA), which elute in a single peak
containing shoulders in systems described previously. The gradient system was
composed of 75 mM KH2 PO 4 (buffer A) and acetonitrile containing 600
mM acetic acid (buffer B). The starting conditions were as follows: column
temperature, 35°C; flow rate, 0.5 ml/min; 44% buffer B. During the first 80
min, the gradient of buffer B was increased to 50%. At 91 min, the percentage of
buffer B was increased to 70% over 15 min and the flow rate was increased to 1
ml/min over 1 min to elute monounsaturated and saturated long-chain acyl-CoAs.
At 120 min, the percentage of buffer B was increased to 80% to elute very
hydrophobic compounds. At 140 min, the percentage of buffer B was returned to
44% over 5 min and the flow rate was reduced to 0.5 ml/min.
Standards long-chain
acyl-CoA may be purchased from Sigma Chemical Co. (St. Louis, MO), except
22:6-CoA from Moravek Biochemical (Brea, CA).
A global method for the determination of tissue level of all the acyl-CoA as
well as the free CoA has been described (Demoz A et al. J Chromatogr 1993,
635, 251). The principle of that determination is the cleavage of the thio-ester
bond of acyl-CoA after incubation with sodium tetrahydroborate (NaBH4)
and derivatization of the free sulphydryl groups with the fluorescent agent
monobromobimane. The separation of the CoA-bimane adduct is achieved on a
reversed-phase HPLC column. The detection limit is said to be lower that 3 pmol.
A precise determination of the acyl-CoA pool is thus possible with only 50 mg of
fresh tissue.
The acid-soluble CoASH is measured on an aliquot of the aqueous acid extract
prior any cleavage treatment.
Other procedures
There are many reports concerning the determination of acyl-CoA esters using
more complex analytical instruments.
After alkaline hydrolysis of long-chain acyl-CoA esters, the volatile
derivatives of the free fatty acids were analyzed by gas chromatography coupled
with mass spectrometry (Kopka J et al., Anal Biochem 1995, 224, 51).
A direct method for measurement of radioactive acyl-CoA esters by radio-HPLC was
reported (Watmough NJ et al., Biochem J 1989, 262, 261).
Further, liquid chromatography-electrospray ionization mass spectrometry was
used for the determination of acyl-CoA thioesters from bacterial cultures (Dalluge
JJ et al., Anal Bioanal Chem 2002, 374, 835). A direct method for
measurement of individual medium-chain acyl-CoA esters in animal tissues using
the same equipment was also described (Kasuya F et al., Anal Biochem 2004,
325, 196).
A highly sensitive method for the quantitation of long- and very-long-chain
fatty acyl-CoA using liquid chromatography combined with electrospray ionization
tandem mass spectrometry has been reported (Haynes
CA et al., J Lipid Res 2008, 49, 1113).
