Fatty acyl CoA analysis

ANALYSIS OF

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).
A review of acyl-CoA analysis by mass spectrometry focuses on mammalian samples and long-chain analytes (i.e. palmitoyl-CoA) has been released (Haynes CA, Biochim Biophys Acta 2011, 1811, 663). It particularly reports of streamlined methodology, quantification and improved recovery.