Isolation of Pyruvic and AI pha-Ketoglutaric Acids from Blood and Tissues in the Presence of Carbon44 Acetate ELLING KVAMME and LEON HELLMAN Division o f Physics and Biophysics, Sloan-Kettering lnstitute for Cancer Research, N e w York 21,
When existing methods were used to separate pyruvic and a-ketoglutaric acids from biological materials as the 2,4-dinitrophenylhydrazones in the presence of sodium acetate-1-carbon-14, the isolated keto acids were contaminated with labeled acetate. The keto acids were separated from the labeled acetic acid by (1) forming their bisulfite addition complex and extracting the acetic acid with ether and (2) vacuum distilling the 2,4-dinitrophenylhydrazones formed from the keto acids derived from step 1. Almost all the contaniinatiiig acetic acid was removed and only 0.05% of acetate-lcarbon-14 was present in the isolated keto acids. Recoveries of pyruvic and a-ketoglutaric acids from blood were 66%. U R I S G a study of the incorpoiation of labeled acetate into the keto acids of liver, it n a s found that the specific activities of pyruvic and a-ketoglutaric acids were unreasonablJhigh when these substances were isolated by existing methods involving the paper chromatographic separation of the 2,4dinitrophenylhydrazones of the keto acids (?-9, 11) -4s it seemed possible that the labeled acetate precursor was contributing to the high specific activity, sodium acetate-1-carbon-14 was added to five samples of blood and the keto acids a e r e then isolnted. From 25 to 407, of the added acetate radioactivity appeared in the isolated keto acid hydrazones and was responsible for the simulated labeling of these rompounds. In order to evaluate the behavior of the keto acid hydrazones and the labeled acetate during paper chromatography, acetatel-c:ilbon-14 was applied with the hydrazones to paper in a butyl drohol-ethyl alcohol-ammonia solvent system (3-5). The data whirh appear in Table I indicate that acetate-1-carbon-14 spread over a large area and Contaminated most of the paper Additional attempts to separate acetate from the hydrazones by e\traction with different solvents, and by precipitation of acetate with heavy metals in the presence of carrier failed t o yield satisf:tctory results. Recrystallization in the presence of carrier acetate was not practical because of the small quantity of hydrazone available. Steam distillation to remove acetate, or any othrr procedure involving elevated temperatures, was unsuitable, because the keto acids were destroyed.
Table I. Paper Square 1
2 3 4 5 6 7
Distribution of Labeled Acetate on Paper Chromatogram Yo Acetate-lRi 0-0 09 0 090-0 178
0 178-0 0 267-0 0 336-0 0 445-0 0 535-0 0 624-0 0 713-0 0 802-0 0 890-0
267 336 445 535 624 713 802 890 980
Carbon-I4 per Paper Square 14.2 12.7 12.1 12.0 21.4 6.9 4.0
2.7 6.8 5.3 1.9 11 100.0 A known amount of acetate-I-carbon-I4 in aqueous solution was applied t o l a 2-cm. wide Whatman KO.1 filter paper strip, which was chromatographed (3-5) overnight a n d cut into 2 X 2 cm. squares Each square was disintegrated in 10% sodium carbonate solution, which was then transferred to a planchet a n d counted. 8
9
10
N. Y.
I n order to make use of the great sensitivity of the methods for the separation of the keto acids as hydrazones by paper chroniatography, especially as compared with other procedures (8, IO). they were modified and it was shown that unlabeled pyruvic and a-ketoglutaric acids could be separated from acetate-lcarbon-14 with only negligible traces of radioactivity remaining in the isolated keto acids. EXPERIMENTAL JIETHODS AND RESULTS
Reagents. Solution I. This contains 0.6024 gram of sodium pyruvate, 1.0267 grams of sodium acetate, and 3.48 X lo8 disintegrations per minute of sodium acetate-1-carbon-14, in 25 nil. of water. 2,&Dinitrophenylhydrszine, saturated solution in 2.Y hydrochloric acid. Aletaphosphoric acid, 5% freshly made aqueous solution. Sodium pyruvate, minimum purity 96%. a-Iietoglutaric acid, melting point 113-18" C. Sodium bisulfite. 1 s . Sodium hydroxide, saturated solution. Sodium hydroxide, 1 s . Sodium hydroxide, 0.01S. Hydrochloric acid, concentrated. Hydrochloric acid, 2 s . Sodium carbonate, 10% aqueous solution. Ethyl acetate. Ethyl ether.
Table 11. Isolation of Keto Acid Hydrazones from Blood in Presence of .Acetate-l-Carbon-14 Blood Sample Acetate-Icarbon-14 added (DPJI) Residual activity In "hydrazones" yo Acetate-Icarbon-I4 remo\-ed
Carrier-Free Acetate 1 2 3 696,000 696,000 696,000
Acetate-40
hlg. Carrier 5 6 696,000 279,000 279,000 4
1.595
1,;''s
1223
970
0
0
99.8
99.8
99 8
99 H
100
ion
Six 10-ml. samples of blood containing acetate-I-carbon-14 were deproteinised with 50 ml. of metaphosphoric acid. The complete, revised procedure was carried out and the dry residue of keto acid 2,4-dinitrophenylhydrazones obtained after reiiioving the ethyl acetate, was dissolved in 5 ml. of 10% sodium carbonate. Aliquots of 0.2 ml. were plated. Samples 1 t o 4 were shaken four times with three volumes of ether, samples 5 and 6 shaken five times with five volumes of ethyl ether. All radioactivity measurements w e x made with probable error of leas t h a n 2%.
I n the revised method, the tissue extract, deproteinized with metaphosphoric acid, is neutralized and treated with sodium bisulfite a t 37" C. in order to form the bisulfite addition products of the keto acids. The solution is then acidified and extracted with excess ether in order to remove acetic acid. The keto acid bisulfite addition products are then dissociated with sodium carbonate and the free keto acids are reacted with 2,4dinitrophenylhydrazine in acid solution. The dinitrophenylhydrazones are extracted with ethyl acetate, which is then shaken with 10% sodium carbonate; the keto acid 2,4-dinitrophenylhydrazones pass into the alkaline layer. Back-washing with ethyl acetate removes most of the remaining 2,4-dinitrophenylhydrazine.The sodium carbonate solution is acidified and the liberated keto acid 2,4-dinitrophenylhydrazones are extracted into ethyl acetate. The ethyl acetate solution is evaporated to dryness a t low temperature in vacuo to remove any residual volatile acid 1995
1996
ANALYTICAL CHEMISTRY
14. As shown in Table 11, 99.8% of added labeled acetate can be removed from Pyruvic Acid Added to Blood a-Ketoglutaric Acid Added to Blood blood by the revised proceBlood Amount Amount Blood Amount Amount dure in the absence of carsample, added, recovered, Recovery, sample, added, recovered, Recovery, rier acetate. When carrier ml . r Y '% ml. Y % 10 121.8 79.2 65.0 10 172 107.5 62.5 acetate is present 99.8 to 100% 70.2 10 86 10 60.9 42.7 58.4 67.9 is removed. 10 24.4 16.9 69.3 10 34.4 22.4 65.0 63.8 10 17.2 10 12.2 7.8 11.4 66.0 The over-all r e c o v e r y of The pyruvate or a-ketoglutarate was dissolved in 1 ml. of saline and added to whole blood. The complete, pyruvic and a - k e t o g l u t a r i c revised procedure was followed; after final ethyl acetate distillation, the residue was made up to volume and the acids was tested by adding recovery measured colorimetrically, with suitable correction for the blank. these substances t o w h o l e blood and then carrying out the complete, revised proand to obtain the 2,4-dinitrophenylhydrazones. Further separacedure up to the final step of the chromatographic separation tion of the hydrazones is carried out on buffered silica gel columns of the hydrazones. A4s shown in Table 111, approximately (1) or by paper chromatography ( 4 , 5, 8, 11). Since pyruvic 66% of the added keto acids were recovered by this procedure acid hydrazone resolves itself into two zones on paper, both and the recovery after chromatographj is almost 100% of this zones were combined in all measurements. The revised method incorporates two new steps for removal of amount (1, 11). radioactive acetate: extraction of the acidified bisulfite addition complex of the keto acids with ether and evaporation of the ethyl Recommended Method. The blood or homogenized tissue is acetate solution of the appropriately washed 2,4-dinitrophenylimmediately deproteinized with a fresh solution of 5% metahydrazones to dryness a t low temperature under vacuum. The phosphoric acid and centrifuged. The supernatant solution is method was subjected to the following tests for efficiency of decanted, neutralized with a saturated solution of sodium removal of labeled acetate and recovery of keto acids. hydroxide, back-titrated with 2 5 hydrochloric acid to pH 6, and warmed for 10 minutes a t 37" C. with 1M sodium bisulfite solution in excess of the quantity of keto acids present. Sufficient The effect of acidity on the removal of labeled acetate was ice-cold concentrated hydrochloric acid is addcd t J bring the determined by incubating 6-ml. portions of hydrochloric acid of solution to a t least 0.5M with rekpect to acid; the solution is differing concentration together with 2 ml. of solution I (279,000 then extracted four times with three volumes of ethyl ether. The disintegrations per minute) plus 2 ml. of 1M sodium bisulfite a t aqueous layer is treated with 10% sodium carbonate to p H 9, 37" C. for 10 minutes in each of four flasks. The flasks were then neutralized with ice-cold concentrated hydrochloric acid and warmed for 15 minutes a t 38" C. with excess fresh saturated extracted eight times with 20 ml. of 10% sodium carbonate. solution of 2,4-dinitrop henylhydrazine in 2N hydrochloric acid. Duplicate 0.2-ml. aliquots of the residue were plated from each The hydrazones are extracted four times with equal volumes of flask, and the data indicated that the use of 2 5 hydrochloric acid ethyl acetate. The ethyl acetate layer is extracted four times with half the volume of 10% sodium carbonate and the excess promoted optimal removal of labeled acetate (74%). 2,4-dinitrophenylhydrazineis removed from the aqueous layer The effect of number of extractions and quantities of ether by back-washing with one third the volume of ethyl acetate. used on the removal of labeled acetate from the acidified bisulfite This sodium carbonate extract is acidified with ice-cold concenaddition complex of the keto acids was tested with from 4 to 8 trated hydrochl~ricacid and the 2,4-dinitrophenylhydrazonesof the keto acids are extracted by shaking three times with half extractions with 2 to 10 volumes of ether. Four ether extracthe volume of ethyl acetate. The ethyl acetate is removed tions with portions five times the volume of the aqueous layer under reduced pressure a t 30" C. A dry ice trap is used to comremoved 98% of the radioactivity in the presence of carrier acepletely recover any acetic acid in the distillate. tate. The same operation with three volumes of ether removed The residue is dissolved in a very small volume of ethyl ether or ethyl acetate and carefully applied to the filter paper to be 89 to 90% of the radioactivity in the absence of carrier acetate. chromatographed. The spots are cut out and dissolved (6, 11). The additional amount shaken out by eight extractions with as The quantities are estimated colorimetrically in a spectrophotommuch as 10 volumes of ether is negligible. The omission of careter a t 445 mfi by comparing with a suitable standard curve; rier acetate permits the specific activity of the undiluted acetate the radioactivity is determined by plating an aliquot on a planchet of 4.9 sq. cm. area on lens paper and counting in a to be easily obtained by shaking the ether with 1 S sodium hywindowless flow-gas counter. The entire procedure should be droxide to remove acetic acid, acidifying the alkaline solution carried.out without delay, preferably in 1 day, to ensure good with hydrochloric acid, and then condensing the acetic acid after recoveries. steam distillation. I n order to determine the radioactivity of the acetic acid which As an alternative to extraction with ether, vacuum distillation was removed, the ethyl ether extract from the first extraction procedure and the distillate from the evaporation of the ethyl a t low temperature (below 40" C.) was tried in order to separate acetate are shaken four times with small volumes of 1N sodium contaminating acetate from the keto acid bisulfite complex. hydroxide. (The ethyl ether extract also contains the major When the volume was reduced to one fifth of the original value, portion of fumaric, succinic, malic, and citric acids.) The sodium hydroxide layer is acidified with hydrochloric acid and 90% of the activity was removed and by continuing to complete steam distilled (Duclaux). The volatile acids in the collected dryness, 97.8% was removed; this procedure is not suitable for fractions are titrated by 0.01N sodium hydroxide and an aliquot routine use, as it is much more time-consuming than the ether is counted as described above. extraction method. After the first new step in which carbon-14-acetate is partially DISCUSSION removed from the keto acid bisulfite addition products by exThe data show that the method of four extractions with three traction with ether, the keto acid 2,4-dinitrophenylhydrazones volumes of ethyl ether, after warming with sodium bisulfite, are formed and are then extracted with ethyl acetate, taken up removes 99.8% of added radioactive acetate; the recovery of in sodium carbonate, re-extracted with ethyl acetate, and subpyruvate and a-ketoglutarate is about 66%. If carrier acetate jected to vacuum distillation a t lorn temperature in order to is added and the four extractions with five volumes of ether are remove the remaining carbon-14-acetate as completely as poscarried out, no activity can be detected, but the recolery of sible. Vacuum distillation alone was 99% effective in removing keto acids is reduced to 40%. acetate-1-carbon-14, either with or without carrier, from the It is an advantage to avoid addition of carrier acetate, so that keto acids hydrazones isolated from blood. Repeated distillathe specific activity of the radioactive acetate can be estimated. tions, either with or without carrier acetate, proved to be of When the total radioactivity present as acetate has been delittle value in removing the remaining traces of acetate-l-carbon-
Table 111. U s e of Revised M e t h o d for Recovery of Pyruvic Acid and a-Ketoglutaric Acid from Whole Blood
1937
V O L U M E 26, NO. 12. D E C E M B E R 1 9 5 4 termined as described, any incorporation of activity into the total keto seids less than 0.2% of that of acetate is not significant and probably represents contamination. After paper cbromatography more contaminating acetate is removed, and activity in the keto acids greater than 0.05% of the acetate is significant.
JLLU,
U .v.,
UY
...
DULLIS,
.....
R. H.. Proc. Natl. Acad.
! -.._ P"t+.* v. . TI ..y..lI.I. n.".L-., _-_"_., _"_.
(1953).
FT U...lI..a-+ (2) Ruseh, I.., 196, 717 (1952).
(1949). (4) Ibid., 164,792 (1949). (5) El Hawary. M. F. S.,and 340 (1953).
Thompson, R. H. S.. Bioohem. J., 53.
(6) Friedmann. T. E., and Haugen, G. E., J . B i d Chem.. 147, 415 (1943). 157, 673 (1945).
The authors are grateful t o Harold Kammen and Michael G. Fusillo far technical assistance and t o Guilio C. Perri, R. S. Rmenfeld, and T. F. Gallagher for invaluable advice.
u. s., 39,754
Csvdlini. D., Frontali. N., snd Toschi, G., Natuie, 163, 568
(7) . . Friedman". T. E., Hawen. G. E., and Kmieeirtk. T. C.. Ihid.,
ACKNOWLEDGMENT
CITE0
(3)
SCi.
I
Ph' -..m~.
(8) Hoekenhull, D. J. D.. Hunter, G. D.. and Herbert, M. W., Chemishy & Zndustry, 1953, 127. (9) Koepsell, H. J., and Sharpe, E. S., Avch. Biochem.. 38, 443
,I
,10101
(10) Phares. E. F.. Mosbltch. E. H.. Denison. F. W.. Jr.. and Carson. S. F., ANAL.Cnm., 24, 660 (1952). (11) Seligson. D.. and Shepiro. B., Ibid., 24, 754 (1952). Riosiveo for review February 25, 1954. Accepted August 25, 1954. This inveatigation WBS suppmted in part by Department of the Army Contract DA-49-0fl7-MD236. E. Kvamme is the recipient of a Sloan Stipend for Advanced Study and a grant-in-aid from the Univemity of Oslo. Norway.
CRYSTALLOGRAPHIC DATA
89. 2,6-Dinitrotoluene (2,6- DWT) Contributed by WALTER C. MCCRONE, Armour Rerearch Foundation of Illinois In and J O H N H. ANDREEN, E. 1. du Pont do Nemoi~ r r Co., Inc., Wilmington, Del CHa
Structural Formula for f,CDinitrotoluene, Form I TXCELLENT oryaais
Well-formed crystals of either I or I1 can be recrystallized from thymol on a microscope slide. The normal product from fusion is 11, although a carefully supercooled melt will nucleate t o give 111. This very unstable farm transforms almost instantaneously, however, to form 11, which, in turn, can be seeded with I and slowly transformed to the stable form I.
or ~,o-umn,~ro~oiuene(i~ can ~e onmined
h by slow cooling of a hat ethyl alcohol solution with con-
C
tinuous agitation. Benzyl alcohol and thymol are good solvents for recrystallization on a microscope slide (Figure 1). Crystals prepared in this way are orthorhombic and show the forms: prism (1101, basal pinacoid { O O l ) , brachy pinacoid (OlO), and braeh ydome i O l l ) . .. 2,6-Dinitrotoluene has a t least three crystal forms .DO^ morp:ha). Form I11 is apparently unstable a t d l temperature s from.,,.. I,ya(11) is stable above about 40" C. and 2,6-dinitrotoluene(I) is yuALA,,uLayuL=
yv
I.I-._
Y..YY.VUY.YI..
. . . . . . .
Figure 2. Orthographic Pmjeotion of Typical Crystal of 2,CDinitrotoluene
..
. . . . . . ...................
Slide
CRYSTALMORPHOLOGY (I.rystal Sp!er+ OLthorhombic. . - . Worm and Habit. Crystals tram ethyl alcohol are tablets and rodsandshowtheprism (IlO),thebrachypinacoid (010),andthe brschydome { O l l } . Many crystals from thymol on a microscope slide show the basal pinaooid (001)in addition to theaboveforms. AxialRstio. a:b:c = 0.571:1:0.53~. Interfacial Angles (Polar). 110 A 110 = 59' 26'; 011 A Oil = 56" 42'.
