Estimation of lactic acid in biological material by oxidation with ceric sulphate.

FROMAGEOT & DESNUELLE [1935] proposed a method of estimation of pyruvic acid based on the oxidation of x-ketonic acids by ceric sulphate. The value of the method is diminished by the fact that numerous substances commonly encountered in biological material, e.g. lactic and citric acids, also react with ceric sulphate. The interference by lactic acid is not serious so long as the amount of the latter is small compared with that of the pyruvic acid present and so long as the temperature at which the reaction is carried out is kept low (e.g. approx. 00). Citric acid is attacked by ceric sulphate even at low temperatures and its possible presence must be taken into account in any estimation of pyruvic acid by ceric sulphate oxidation. Whilst investigating the magnitude of the error in pyruvic acid estimations caused by the presence of lactic acid in tissue extracts, we found that at relatively high temperatures, e.g. 500, lactic acid is oxidized quantitatively by ceric sulphate to acetaldehyde. Analysis showed that the reaction obeyed the equation CH3.CHOH.COOH+ 2Ce++++= CH3.CHO + C02+ 2H++ 2Ce+++. Lactic acid could be estimated with accuracy by allowing it to react with an excess of standard ceric sulphate solution at 500 for a suitable time and titrating the excess Ce++++ with standard ferrous ammonium sulphate solution. This procedure is of little value when applied to biological material owing to the large errors introduced by the presence of other substances attacked by ceric sulphate under the conditions employed. Estimation, however, of the acetaldehyde produced by the oxidation of lactic acid showed that its amount was not materially affected by the presence, in the lactic acid solution, of substances commonly encountered in tissue extracts, so long as excess ceric sulphate was used. The method we have adopted for the estimation of lactic acid consists of the oxidation of the substance by ceric sulphate to acetaldehyde, followed by estimation of the latter by absorption in sodium bisulphite solution as in the well-known method of Friedemann et al. [1927]. We find that the presence of glucose, fructose, starch and a variety of other substances does not interfere with the lactic acid estimation so long as these are not present in abnormally large concentrations. Hence it is unnecessary to remove these substances from tissue extracts before making lactic acid estimations. An estimation of lactic acid in blood requires only treatment with trichloroacetic acid, an aliquot of the filtrate being treated at once with ceric sulphate solution. The same procedure is carried out with tissue brei, e.g. liver or brain.