Preparation and analyses of CoA derivatives

Coenzyme A (CoA) derivatives of bile acids were prepared [33,34] by a modification of the mixed anhydride procedure for the synthesis of palmityl CoA. A yield of 70% was calculated from the quantity of cholylhydroxamate formed after treatment of the CoA derivative with hydroxylamine. By modification of this procedure and purification of the products by chromatography on Sephadex LH-20, aqueous solutions of CoA derivatives of cholic, chenodeoxycholic, deoxycholic, and lithocholic acids were pbtained, frozen at - 70°C, and shown to be free from hydrolysis for several months [35]. 

Separation of bile acid-CoA derivatives from unreacted coenzyme A can be effected by high-performance liquid chromatography. With a radial compression C,g column containing 5-/tm particles, coenzyme A was eluted with or immediately following the solvent front with a solvent system of 2-propanol/50 mM phosphate buffer (pH 7.0) (160/340) and a UV-detector set at 254 nm. Capacity factors for the above CoA derivatives are 1.50, 3.50, 4.18 and 9.50. CoA derivatives of comparable 5a-bile acids, purified and separated in a similar manner were eluted just prior to the comparable 5)8-derivative [36]. CoA derivatives of 5jS- and 5a-cholate and de-oxycholate were separable with this solvent system. 

A mechanism for hepatic formation of amino acid conjugates of bile acids developed from early investigations of Siperstein and Murray [37], Bremer [38], and Elliott [39] is summarized as follows  

Bile acid -f- ATP CoASH bile acid-SCoA + AMP PPj (1) 

Bile acid-SCoA -(- amino acid 5= bile acid-NH-amino acid -I- CoASH (2) 

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