Florijn, E.

Marken Lichtenbelt, H. van

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Pyruvate content of blood and phosphorylation of thiamine in diabetic coma

Utrecht University Repository

VOL. 8 (1952) SHORT COMMUNICAT IONS,  PREL IMINARY NOTES 349 PYRUVATE CONTENT OF BLOOD AND PHOSPHORYLAT ION OF THIAMINE IN DIABETIC COMA by H. VAN MARKEN L ICHTENBELT  AND E. FLORI JN  Department o/Medicine and Laboratory o/Physiological Chemistry, The University, Utrecht (Netherlands) According to MARKEES AND MEYER 1 an increased content of pyruvate is found in the blood of patients in diabetic coma. It  decreased by intravenously injecting thiamine pyrophosphate, but not, however, by injecting thiamine. The authors concluded that in this condition phosphorylation is inhibited. We have found, however, that in these investigations the pyruvate determinations, which were performed according to the method of FRIEDEMANN AND HAUGEN z, have been carried out in such a way that pyrnvate was determined together with acetyl acetate. Overnight storage of the deproteinized blood in the refrigerator, as prescribed by FRIEDEMANN AND HAUGEN and as had been done by MARKEES AND MEYER, did not appear to be efficacious for removing the acetyl 60 --,% 5O ~.~ x\ x 5 ~|  450 300 150 1 Hours Fig. z. Diabetic coma treated with several insulin injections. Abscissa: hours after Rrst injection. × . . . . .  × "pyruvate" ,  without heating in boiling water bath • . . . . .  • pyruvate, after 5 rain in boiling water bath [] [] blood sugar 0 0 alkali reserve . /2 \  / \ ~- - -~/ "  " \ . \  l . / / " /~"  ~ \'\'~, o J 6 b I~ I~ Ho~/~ Fig. 2. Thiamine pyrophosphate in blood. Abscissa: hours after injection. 0 - - - - - -0  injection of ioo mg thiamine pyro- phosphate in normal subject ×- - - -X  injection of 6o nag thiamine in pa- tient in diabetic oma acetate. All acetyl acetate is decomposed, however, by placing the deproteinized blood in a boiling- water bath for 5 minutes. The pyruvate content is not affected by this treatment. After the conclusion of our experiments a note by MARKEES $ appeared, showing that also he had independently arrived at the same results. 350 SHORT COMMUNICATIONS, PRELIMINARY NOTES VOL. 8 (1952) Fig. I shows the results of the t reatment  with insul in of a pat ient  in diabetic coma. In  th is  exper iment  FRIEDEMANN AND HAUGEN'S method was each t ime appl ied to two samples of deprote- inized blood, one of which had been stored for 24 hours in the refrigerator, while the other had been placed in a boi l ing-water bath  for 5 minutes.  This  figure proves that  the pyruvate  content  of the blood was not higher than  would have been found in a normal  subject in the same state of muscular  act ivity.  The quest ion of the d iminished phosphory lat ion remains.  We found the th iamine pyrophosphate content  of the blood in diabetic coma had not  decreased. When th iamine was administered to a pat ient  in diabetic coma by int ravenous injection the th iamine  pyrophosphate  content  of the blood slowly increases, just  as happens  in normal  subjects, at ta in ing a max imum several hours after the injection. In jected th iamine pyrophosphate disappears from the blood of normal  persons as well as from that  of the  pat ients  in 3 ° to 6o minutes.  This  init ial  rapid decrease of the  th iamine pyro- phosphate  content  was in both cases followed by a slow increase, similar to the increase obtained after th iamine injection (Fig. 2). The explanat ion can obviously be found in the rapid decomposit ion of injected th iamine pyrophosphate by the p lasma phosphatase,  followed by phosphory lat ion of the th iamine,  thus  formed, by the blood corpuscles 4.Hence we believe that  in diabetes neither the breakdown of pyruvate,  no1 the phosphorylat ion of th iamine are affected. Ful l  detai ls will be publ ished. REFERENCES 1 S. MARKEES AND F. W. MEYER, Schweiz. reed. Wochenschr,, 79 (1949) 931. 2 T. E. FRIEDEMANN AND G. E. HAUGEN, J. Biol. Chem., 147 (1943) 415 • 3 S. MARKEES, Experientia, 7 (1951) 314 • 4 H.  G. K. WESTENBRINK AND E. P. STEYN-PARV~, Inter. Rev. Vitamin-R~search, 21 (195o) 461. Received January  I9th, I952 THE ENZYMIC BREAKDOWN OF DEOXYRIBOSENUCLEIC ACIDS by J. D. SMITH AND ROY MARKHAM Agricultural Research Council Plant Virus Research Unit, Molteno Institute, Cambridge University (England) From recent work on deoxyribosenucleic a ids (DNA), including the isolation of nucleoside- 5 -phosphates  by successive t reatment  of DNA with deoxyribonuclease and phosphodiesterase 1,~,it seems probable that  the major i ty  of the nucleotides are joined through phosphate  ster l inks on carbon atoms 3' and 5'. Deoxyribonuclease d polymerises DNA, l iberat ing some f ragments  which readi ly dialyse through Cellophane, and others which have been believed to be non-dialysable. Electrometr ic t i t rat ion has shown that  about  1 secondary phosphoryl  dissociat ing group is l iberated for every 4 nucleotide residues 3. No free phosphate  is formed. Some of the polynucleot ide products  must  therefore be small ,  but  while their  part ia l  separat ion has been effected by electrophoresis 4 the analyses obtained on these preparat ions were not  adequate to permit  the identif ication of the  const i tuents  with any  certainty.  Using the paper chromatographic  and electrophoretic techniques which we have developed for the separat ion of polyr ibonucleotides 5, we have isolated and identified some of the enzymic breakdown products  from DNA. Herr ing sperm DNA e (20 mg/ml)  was digested with deoxyribonuclease ~ (2o/~g/ml) in o.oo 5 M MgSO 4 at pH 7 for 18 h. A trace of CHC13 was present o prevent  bacterial  growth. This  digest was dialysed into water, al lowing some of the smaller f ragments  to escape, and the dialysate concentrated in vacuo. Pre l iminary fract ionat ion of the dialysate was possible by paper chromatography in 70% iso- propanol-water (v/v), with NH 3 in the vapour phase, but  separat ion into sharp bands was difficult. 

Pyruvate content of blood and phosphorylation of thiamine in diabetic coma

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