Cxxxi. Pyruvic Acid as an Intermediary Metabolite in the Brain Tissue of Avita- Minous and Normal Pigeons

of results (averages). R.P. R.P.+V. L. L.+V. Initial 2 hours 2 hours Diff. 1 hour 1 hour Diff. Residual. Normal (1) 1 18 Avitaminous (5) 1-17 2-12 1-69 0*43 Lactate. Normal (6) 0-92 2-32 Avitaminous (11) 6-48 4-89 1.59 L. +Fl. L. L.+V. L.+Fl. +V. L.+ L.+GI. 2 hours 2 hours Diff. 2 hours 2 hours Diff. lodo. L. + G. +Iodo. Lactate. Normal (6) 3-33 3-19 7-72 3-18 5-67 Avitaminous (11) 9-28 5-58 3 70 5-56 3 90 1-66 Results in Table I are the mean of duplicate estimations; they agree within ±2 % for L. (avit.), (±0-01 ml. iodine for the equivalent of 1-0 ml. bottle contents). For smaller amounts of pyruvate the error is proportionately greater. V. =in all cases, 2y crystalline vitamin B1 hydrochloride per Barcroft bottle [Kinnersley et al., 1933]. Substances combining with bisulphite are present in very small amounts in normal brain and increase slightly during respiration in lactate (not more than 04 mg. per g.). The amounts formed with avitaminous brain are of a different order (ten times as much); they decrease 39 % with added vitamin. A comparison of experiments in Ringer-phosphate only, with those in lactate show a slight increase of B.B.S. with lactate in normal, but a large increase for avitaminous brains. Hence lactate is essential for the formation of the B.B.S. which are decreased in amount by added vitamin. Before proceeding further, it is necessary to know how much of the B.B.S. is really pyruvic acid. The method of Clift and Cook [1932] of heating in alkali to remove substances other than pyruvic acid did not prove satisfactory in our hands, and could not be so certain as isolation of the 2: 4-dinitrophenylhydrazone [Case, 1932]. Case's method involves the following steps: 1. Addition of 2:4-dinitrophenylhydrazine to the trichloroacetic acid solution. 2. Extraction of mixed hydrazine and hydrazones with ethyl acetate. 3. Neutralisation with CaCO3. 4. Evaporation and extraction with toluene. 5. Extraction with NasCO3. 6. Acidification and extraction with ethyl acetate. 7. Evaporation to dryness. 8. Estimation in solution in alcoholic potash. This has been modified as follows. Stage 1. Same; 5 ml. 1 % hydrazine in 2N HCI added to 20-0 ml. trichloroacetic acid solution. PYRUVIC ACID IN METABOLISM OF BRAIN 919 2. Same; at least 3 extractions, and often more. 3. Successive extractions with alkaline phosphate, 1/3 vol. The first extraction with saturated alkaline phosphate (Na), and subsequent with M/15. 4. Acidification with HCl and extraction with ethyl acetate. 5. Repeat stage 3. 6. Repeat stage 4, making acid to pE 2-5 approx. 7. As Case. 8. As Case. We found that our extracts did not give good results by Case's method unmodified. Since the pure hydrazone of pyruvic acid was found to be completely extracted by alkaline phosphate we proceeded as in stages 3 and 4, continuing the extraction with alkaline phosphate until fresh extracts were colourless, or showed only a faint brown-yellow colour, due to traces of hydrazine. With this exception other stages were repeated until colourless. The trace of hydrazine carried over into 4 is removed almost completely in 5 and 6. The combined ethyl acetate solutions from 2-3 bottles (equivalent to 200-300mg. tissue) are conveniently made up to 5-6 ml. 1-2 ml. (Ostwald pipette) can be evaporated in a small measuring flask to 0 1 ml., cooled and made up to the mark with the alcoholic potash. Standards containing 0 5 mg. per 1 0 ml. ethyl acetate have been used for comparison (_0-15 mg. pyruvic acid). The 0.1 ml. ethyl acetate left assists solution in the alcoholic potash and minimises the risk of hydrolysing the hydrazone. It appears to have no influence upon the determination provided that the estimation is performed reasonably soon. The method has the great advantage that heating is always avoided up to stage 6, and if necessary after this by evaporation in vacuo. Hence unstable hydrazones such as that of oxaloacetic acid [Clift and Cook, 1932] should be detected more easily. Stages 2-6 for 4 estimations take approximately 4 hours. Stage 7 reaches a high accuracy with 0 5 mg. or even less of hydrazone. Stages 2-6 give a good recovery of pyruvic acid 2:4-dinitrophenylhydrazone added at stage 2 to the combined ethyl acetate extracts. Examples are given in Table II. Table II. Recoveries. 1. Pyruvic acid 2:4-dinitrophenylhydrazone added at stage 2. Final ethyl acetate phase made upto 6-0ml.approx.and20 ml.takenfor analysis in all except c (3.0 ml.). 1.0 mg. =0-328mg. pyruvic acid. Added Found Corr. mg. mg. mg. (a) 1-0 1-02 1.0 (b) 1.0 1-02 1.0 (c) 1.0 1-03 1.0 (d) 1-5 1-49 1-48 2. Pyruvic acid added at stage 1. Added Found Corr. mg. mg. mg. (e) 0-492 0 494 0-488 (f) 0-492 0-496 0 490 (g) 0-984 0-984 0-981 (h) 0-984 0-960 0 957 As stated previously a trace of hydrazine accompanies the hydrazone, making a difference of approx. 0.01 mg. hydrazone (0.003 mg. pyruvic acid) per 1.0 ml. of final phase taken. This can be allowed for as a blank, if desired. Our slight modification is presumably more specific in its extraction than the Na2CO, stage, but we have not investigated this. Table III gives the results of estimations by the modified Case 2: 4-dinitrophenylhydrazone method upon solutions from some of the experiments given in Table I. The experiments are given in order and probably increase in accuracy; Biochem. 1934 XXVIII 59 920 R. A. PETERS AND R. I. S. THOMPSON Table III. Substrate lactate. Pyruvic acid, estimated by 2:4-dinitrophenylhydrazone method. mg./g. Diff. L. L.+V. 02 tL./I Exp. L. L. + V. (L. + V.) L. %* %* uptake mg. P.A. Remarks 554 3-32 1*66 1-66 81 72 1345 815 M.P. hydrazone low, 1930 557 2-90 1-68 1-22 67 63 962 790 M.P. 216° 558 3-67 1-57 2*10 84 69 1010 480 M.P. low, 2040 560 3-30 1-37 1*93 68 58 1115 580 (No pyrophosphate) 569 3-31 1-79 1-52 102 88 1162 765 580 3-88 1-89 1*99 90 82 1020 515 Average 3 40 166 1i73 82 72 [102 657 * % of B.B.S. estimated as pyruvic acid 2:4-dinitrophenylhydrazone. Exp. 590. Samples ofavitaminous cerebrum in bottles as follows: 4 contained L. only, 4 L. + V. AU were treated similarly, but two of each were removed from the bath after the preliminary shaking period, cooled and treated with trichloroacetic acid. The remainder were shaken 1 hour.