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The amides of glutamic and aspartic acids play an important role in cell metabolism and nitrogen transport in multicellular organisms. In addition, they are common constituents of proteins. However, the amide group is acid-labile and thus is completely destroyed under the strongly acidic conditions required to hydrolyse proteins. The amide group is likewise hydrolysed in any procedure involving acid-catalysed esterification, although not necessarily completely. Thus, assay methods for amino acids based on their conversion to volatile derivatives for analysis by gasliquid chromatography (GLC)'J cannot be used directly to assay glutamine and asparagine.
A number of modifications to derivatisation procedures for GLC analysis of amino acids have been described. These are based on the principle of minimising the destruction of glutamine and asparagine under precisely controlled conditions so that conversion of the amides to the corresponding acids is reproducable. Appropriate conversion factors can then be calculated. For example, Hediger et ~1.~ modified the direct esterification procedure of Roach and Gehrke4 by reducing the time and acid concentration required for complete formation of the amino acid n-butyl esters. Samples were esterified for precisely 7 min at which time the ratio of the amide to the acid forms was a maximum at 0.8 for aspartate and 1.8 for glutamate. This method was applied to enzymic hydrolysates of proteins. An analysis of an acid hydrolysate was required to obtain the total amounts of glutamic and aspartic acids.
Collins and Summers also used the procedure of Roach and Gehrke4, and observed that the proportion of glutamic acid converted to pyroglutamic acid depended on time, temperature and acid concentration. However, if these factors were constant, the ratio was independent of the glutamine concentration. By using the same strategy as Hediger et aL3, they were able to determine the amount of glutamine and asparagine with an error of 5% or less.
Young and Desiderio6 converted glutamic and aspartic acids and their amides to the corresponding thiazolinone derivatives and concluded that hydrolysis of the amide side chains was avoided. However, inadequate information was provided to enable duplication of the procedure.
All of the above procedures use indirect means to assay glutamic and aspartic l NRCC No. 24155.
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A sensitive method has been established for the analysis of serum bile acids by gas-liquid chromatography (glc). Bile acids are extracted from 0.5-2 ml of serum and analysed as methyl ester trifluoroacetates following enzymatic hydrolysis of the taurine and glycine conjugates. The method as describe