Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue

Abstract

A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: 1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1M potassium phosphate, pH 6.7, and 2) purification using 2-(2-pyridyl)ethyl functionalized silica gel. Recoveries determined by adding radiolabelled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-or arachidonyl-coenzyme A to powdered rat liver varied from 93% to 104% for tissue extraction and 83% to 90% for solid phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters widely differing in chainlength and saturation.Published methods for the analysis of tissue acyl-coenzyme A content are focused on either short-[1,2], medium-[3] or long-chain acyl-coenzyme A esters [4,5], with the isolation of one acyl-coenzyme A subgroup to the exclusion of others. Recovery of acyl-coenzyme A esters from tissue specimens is often disappointing, with documented recoveries between 30 and 60% [1,5,6]. A general procedure for the isolation of a wide range of acyl-coenzyme A esters, with good documented recoveries from tissues, is presently unavailable.Despite their widely different polarities, we have shown that acylcarnitines (short-, medium-, and long-chain) can be isolated, in a single fraction, from biological samples using organic solvent extraction followed by ion-exchange solid phase extraction (SPE) [7]. This is a highly selective approach, since it combines two orthogonal procedures [8] of isolation: organic solvent extraction and ion-exchange. Extraction procedures for short-chain acyl-coenzyme A esters often use acid precipitation [1,2], but these methods would exclude long-chain acylcoenzyme A esters. Therefore, we investigated extraction procedures originally developed for long-chain acyl-coenzyme A esters, using a mixture of acetonitrile, isopropanol, and aqueous buffer [4]. An SPE anion-exchange column is needed that would be uncharged at pH 7, since elution at high pH would cause hydrolysis of acyl-coenzyme A esters. Finding none commercially available, we ordered custom SPE columns from Supelco, (Bellefonte, PA), packed with 100 mg of 2-(2-pyridyl)ethyl functionalized silica gel. The pKa of these SPE *Corresponding author. Fax: +1 216 368 5162. E-mail address: charles.hoppel@case.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , we analyzed rat heart, skeletal muscle, and liver and we found that the recovery of malonyl-coenzyme A from liver was much worse than for skeletal musc...

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

“…However, most methods only assay either short-, medium-, or long-chain acyl-CoAs (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The best approach for acyl-CoA quantitation, so far, is to measure the target acyl-CoAs from short to long carbon chain length in a single method ( 20,21 ). However, no methodology previously described allows quantitative profi ling of acyl-CoAs for the purpose of targeted and untargeted acyl-CoA analysis.…”

Fatty Acid Chain Elongation in Palmitate-perfused Working Rat Heart

“…A further difference between the two studies concerns the analysis of stable isotope enrichment in malonyl-CoA. In the present work, the 13 C enrichment was determined in malonyl-CoA isolated and specifically detected following solid-phase anion exchange extraction and HPLC separation (17,43). In contrast, in our earlier work (10) the 13 C enrichment of malonyl-CoA was determined indirectly as malonic acid by GC/MS following base hydrolysis of a fraction isolated using an oligonucleotide column.…”

“…Analysis of 13 C Enrichment in Cardiac Acylcarnitines and Acyl-CoAs-Acylcarnitines and acyl-CoAs were extracted from powdered heart tissue by homogenizing the tissue powder first with acetonitrile/isopropyl alcohol (3:1, v/v) followed by 0.1 M KH 2 PO 4 (pH 6.7) and a second 30-s homogenization (17). Acylcarnitines were isolated by strong cation exchange solid phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, and analyzed by UHPLC-MS/MS for content and for 13 C enrichment as described in detail earlier (18,19).…”

“…Acylcarnitines were isolated by strong cation exchange solid phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, and analyzed by UHPLC-MS/MS for content and for 13 C enrichment as described in detail earlier (18,19). Acyl-CoAs were isolated using solid-phase anion exchange columns (Supelco) as described (17). For acyl-CoAs, 500 l of the tissue extract was applied to an SPE column containing 100 mg of 2-(2-pyridyl)ethyl-functionalized silica gel.…”

“…For acyl-CoAs, 500 l of the tissue extract was applied to an SPE column containing 100 mg of 2-(2-pyridyl)ethyl-functionalized silica gel. On-line WAX trapping was used to trap all of the acyl-CoAs, followed by UHPLC reversed-phase chromatography and detection by positive ion multiple reaction monitoring (17,20). The stable isotope enrichment was determined by deconvolution of the isotope patterns using the Isopat 2 algorithm of Gruber et al (21).…”