Further Evidence for Methylatable Precursors of Choline in Natural Materials. ∗

Abstract

In the course of work on choline metabolism in chicks the choline content of several diets was determined by both a colorimetric method and a microbiological method. The colorimetric method was essentially that of Thornton and Browne 1 using reineckate precipitation. Ethanol was used to extract the choline-bearing substances from the original materials. Hydrolysis was carried out with 3% H₂SO₄ and the precipitation was done after neutralization to pH 6.0 with solid Ba(OH)₂ and then to pH 7.5 with 1 N NaOH. The microbiological method was that of Horowitz and Beadle 2 using the Neurospora “cholineless” mutant 34486. The standards as well as unknowns were passed through the adsorption and elution steps. Reproducibility was poor with the Neurospora method on some samples. The results are shown in Table I.

On the purified and semipurified diets the 2 methods were in close agreement. On the practical diet with liver meal and the simplified diet, however, the Neurospora method gave substantially higher values. The simplified diet contained 15% peanut meal.

The value of >0.08% obtained for the simplified diet by the Neurospora method is in essential agreement with that published by McGinnis, Norris and Heuser. 3 The value of <0.04% obtained by the reineckate method is in agreement with a value calculated from the choline contents of the individual ingredients as published by Engel. 5

The higher value obtained by Neurospora on the practical diet containing liver meal might be expected in view of the results of Jukes and Oleson. 6 These authors found that, if a crude aqueous extract of liver was hydrolized with Ba(OH)₂ and then precipitated with reineckate, the resulting filtrate was active for Neurospora.

Jukes and Dornbush 7 observed that dimethylaminoethanol stimulated the mutant Neurospora in a manner similar to choline. Apparently, however, dimethylaminoethanol was not precipitated by reineckate when pure and was only partially precipitated in the presence of choline.