V O L U M E 27, NO. 2, F E B R U A R Y 1 9 5 5 287

quantities of cobalt was tested by running 1 mg. through the procedure and aliquoting the ashed solution of the extract before the final color development; recovery %as 100%. The plot also shows very close adherence to Beer’s law.

The recovery of the method under adverse conditions was tested using commercial steamed bone meal as a convenient ma- terial for analysis. Since this meal contained appreciable amounts of metallic iron as an impurity, it was a more severe test than ordinarily would be encountered. Known amounts of cobalt !+ere added to 10-gram portions (equivalent in ash content to 25 grams of ordinary bone). Thpse were dry ashed and run through the procedure, giving the results indicated in Figure 1. The amount of cobalt plotted was taken as the mean amount found in the meal (2.4 y ) plus the amount added. Average re- covery nas 95%. It may thus be concluded that 5% would be the maximum loss which would be encountered with bone samples up to 25 grams in size; mith smaller samples the loss would be less.

The procedure showed a very high order of specificity (Table I). In order to obtain a true figure for the interference of iron and nickel it 11 as necessary to purify the reagent grade salts of these metals: The required quantities of ferric and nickelous salts were made up in 1 to 49 phosphoric acid media and cobalt impurities were extracted exactly according to the procedure. Following the careful separation of the chloroform and evaporation of the drop floating on the surface, the aqueous residue contained the purified salts. Knonn amounts of cobalt were then added and the usual procedure was follom ed beginning with the extraction step. The purified ferric salt was used immediately to avoid slow reduction to the ferrous state. No appreciable interference oc- curred from quantities as large as 25 mg. of ferric iron and 5 mg. of nickel. Although ferrous iron should not normally be present in an ashed sample, tests were made with 1 mg. of the analytical grade salt (unpurified). The slight positive figure in the absence of cobalt is probably due to the cobalt impurity in the reagent; recovery of added cobalt was 99%. Copper gave no color by itself but reduced the recovery of added cobalt; houever, only a 2% loss occurred with 1 mg. of this metal Larger amounts of copper may be removed by a preliminary extraction with a chloro- form solution of dithizone from the ashed solution of sample

adjusted to pH 2 (6). determination. in the analysis of most biologic materials.

obtained by the procedure, are given in Table 11.

Other metals do not interfere with the Thus no appreciable interferences would occur

Typical cobalt contents of normal bone and tissue samples,

ACKNOWLEDGMENT

The author is indebted to F. L. Hyslop for assistance with the literature search and to D. H. Byers and H. E. Stokinger, under whose direction the work was carried out, for their valuable criticism and review.

LITERATURE CITED

Black, I . A . , Soil Sci., 51, 387-90 (1941). Boyland, E., Analyst , 71, 230-1 (1946). Ellis, G. H., and Thompson, J. F.. ISD. Est,. CHEM., h . 4 ~ . ED., 17, 254 (1945).

Hall, A. J., and Young, R. S., . ~ x . A L . CHEJf., 22, 497 (1950). Houk. A. E. H., Thomas, A. W. and Sherman, H. C., J . Sutri-

fion, 31, 609 (1946).

(19481. Keenan, R. G., and Flick, B. hl., ASAL. CHEX.. 20, 1238-41

Kidson, E. B., and Askew, H. O., S e w Zealand J . S c i . Technol.,

McKnaught, K. J., Analyst, 67, 97 (1942). [bid., 69, 307 (1944). hIcYaught, K. J., &Yew Zealand J . Sci. Technol., 30, 109-15

Maraton, H. R., and Dewey, D. W., Australian J . Ezptl. Biol.

Nichol. W. E., Can. J . Chem., 31, 145-9 (1953). Pascual, J. N., Shipman, W. H., and Wilson, S., .IN.AL. CHEM..

Paulais, AI. R., Ann. pharm.franG., 4, 101-10 (1946). Salteman, B. E., ANAL. CHEY., 24, 1016-20 (1952). Sylvester, N. D., and Lrtmpitt, I.. I-I., J . SOC. Chew!. Ind. (Lon-

Young, R. S., Pinkney, E. T., and Dick. R., IND. ENG. C,HEM..

Waldbauer. L., and Ward, 3. M., Ibid., 14, 727-8 (1942). Welcher, F. J., “Organic Analytical Reagents.” Vol. 111, Yew

Willard, H. €I,, and Kaufman, S., ; ~ s . A L . CHEM., 19, 505 (1947).

B21, 178-89 (1940).

(1948).

Med. Sci., 18, 343-52 (1940). *

25, lh30 (1953).

d o n ) , 59, 57-60 (1940).

-4x.k~. ED., 18, 474 (1046).

York. D. Van Nostrand Co.. 1947.

RECEIYED for rei-iew August 11, 1954. Accepted Sorerriher 1, 1954.

Separation of Alpha-Keto Acid Dinitrophenylhydrazones by Paper Electrophoresis and Their Colorimetric Determination HENRY TAUBER U. S. Public Health Service, University of North Carolina, Chapel Hill, N. C.

This report describes a convenient procedure for the separation and quantitative determination of pyruvic acid and a-ketoglutaric acid. These keto acids are iso- lated in pure state as the 2,4-dinitrophenylhydrazones and separated by filter paper electrophoresis. The separated bands are extracted with sodium hydroxide and the color intensities are measured colorimetrically. The procedure has been applied to the analysis of dif- ferent mammalian bloods.

E T 0 acids form a link betm-een carbohydrate and protein K metabolism. Each keto acid has its specific function. Thus, the determination of the total keto acids in biological ma- terials as practiced in the past is of limited value. A suitable and rapid procedure for the quantitative determination of each of these compounds is of biochemical interest.

Recently several investigators ( I - $ , 8) recommended methods

for the determination of pyruvic acid and a-ketoglutaric acid based on the chromatographic separation of their 2,4dinitro- phenylhydrazones. I n all of these chromatographic methods the hydrazones are separated on large sheets of filter paper with the aid of a combination of solvents and salts. The procedure requires a t least 24 hours. Under those conditions pure pyruvic acid separates into two or three spots (1-4, 8). It has been sug- gested that these spots are the cis-trans isomers and a polymer of the pyruvic acid hydrazone. Paper chromatography, based on this procedure, is not the ideal method for the analysis of a-keto acids.

There is described here an application of filter paper electro- phoresis to the separation of the 2,4dinitrophenylhydrazones of a-ketoglutaric acid and pyruvic acid The separated bands are extracted u ith sodium hydroxide and their color intensities are determined colorimetrically. Commercially obtainable ap- paratus are employed. The procedure has been applied to the analysis of protein-free blood filtrates.

288 A N A L Y T I C A L C H E M I S T R Y

Because the major a-keto acid constituents of the blood are pyruvic acid and a-ketoglutaric acid (the other keto acids are seldom Eee'n and then only in traces), this report deala only with these two keto acids. However, there is a hydrazone obtainable

I is not completely foi J not investigated

from protein-free filtrates of mammalian blood which has a mobility very close to that of rrketoglutaric acid hydrazone but different characteristics.

The present investigation was well in progress when the inter- esting paper by Neish (6) on the separation of a-keto acid dinitro- phenylhydraeones and their polarographic estimation came to the author's attention. Some of his separation experiments have been repeated and extended and a comparative study of the blood of ~everd mammals was made. The polarographic method in which the nitro compounds are reduced in acid solution permits the measurement of very small quantities of keto acids. Ac- cording to Neish (6 ) , however, his method 31-

proof. The polarographic method war in this work.

EXPERIMENTAL

Solutions. 6N hydroohloric acid; 1M sodium carbonate; 1N sodium hydroxide; ethyl acetate, c.P.; 400 mg. of 2,4-dini- trophenylhydraeine dissolved in 40 ml..af 95% ethyl alcohol and 10 ml. of concentrated hydrochloric and, and diluted to 200 ml. with 95% ethyl alcohol; 10% sodium tungstate; 0.67N Sulfuric acid; bmhital buffer of 0.1 ionic strength 155.8 grams of sodium barhital, 38.8.gr;tm of sodium acetate (3HzO),,5900 ml. of dis- tilled water, and ahout 48 ml. of 1N hydrochlonc acid bo make a find pH of 8.5; the final volume is made U@ to 6000 ml.].

Stopcock grease (Nonaq, Fisher Scientific Co.) that is insol- uble in organic solvents was employed. Ordinary stopcock grease is readily carried over during the final extraction of the hydrazones with ether and the grease retains a small quantity of the hydrazones on the line of application.

Purified crystalline sodium pyruvate wa8 used in these investigations (7). The a-keto- glutaric acid (Organic Specialties, he . ) was not further puri- fied. Bath were p~~~~~ , I~ I~ ~ ~~~~~~~ l ~ - ~ ~ l ~ - L ~ ~ ~

phoresis. Stack solutions of of the free acids per 10 ml. of I

and were further diluted with centrations. The hydrazones

Preparation of Pure Hydrazones.

conventional procedure. To 100 ml. of keto acid so1

keto acid or mixtures of keto hydrasine were added. The I 25O C. It was then put into I hydrmones were extracted tl ethvl acetate. Emulsions. if iieine for a few minutes.' 1 were extracted twice with 31 bonate. The combined sadir t.wirn wit,h 2Rml. Dartions o

Prior to the application of the hydrazones the filter paper strips were saturated with barbiturate buffer of pH 8.45. Neish (6) employed 0.05M sodium carbonate a6 the electrolyte. Bar- hiturate buffer, however, offers neater separation and narrower bands. Excess buffer was removed by blotting the strips with sheets of (20 X 20 inches) Eaten-Dickman filter paper No. 320. These sheets were found to he very convenient because of their great ahsorbing capacity. The sheets were re-used many times, as, owing to their unusual thickness (3 mm.) they are durable. The electrophoresis wm conducted a t 23' C.'in a temperature- and humidity-controlled room. This precaution, however, was necessary only during unusually high summer temperatures.

Pure pyruvic acid and a-ketoglutaric acid hydrazones were separated in 2 to 3 hours. Blood pyruvic acid and a-ketoglutaric hydrsaones were separated in 4 to 5 hours. Then the frames containing the strips were removed and dried a t 40' C. in an oven with circulating air supply.

After the strips have been dried the yellow colored hands were cut out, sliced, and laced in centrifuge tubes. The yellow center line or bar whicioften separated between the pyruvic acid and a-ketogluta"e acid hydrazone bars and which appeared close to the a-ketoglutaric acid hydraaone bar was always included with a-ketoplutaric acid hvdrasone (see discussion of component

added to the is and 12 ml.

X). tube

. . , . . . 300

250 -

-

m

3

5200 -

+ 150- 5 100-

L 50 -

Six ml'lliliters of 1M-sodium hydroxide were containing a-ketoglutaric acid hydraaone shrei

I

, IO 15 20 25 30 35 .. .

J 0" !Jg

("Una w "e pure uy prtpu cie(ilir0- the keto acids containing 10 mg. listilled water were prepared daily, distilled water to the desired con- were purified and isolated by the

utiou containing 100 to 1500 y of acids. 5 ml. of 2,4dinitrophenyl-

nixture wa6 kept far 30 minutes a t c 250-ml. separatory funnel and the ree times with 20-ml. portions of formed, were separated by centri- 'he combined ethyl acetate layers bml. portions of 1M sodium car- Im carbonate layers were washed f ethyl ?cet?te, _tfansfcrred.qo an

Figure 1. Calibration curves of hydrazones of pyruvic acid and a-ketoglutaric acid after

separation by paper eleotrophoresis Electrol~te, barbiturate bnffer.,pH 8.45 Time, 3 hours Tempersfure, 23' C.

~ ~.~ ~~

Erlenmeyerflask, &d cooled in an me bath, 'lhen the cold oar- honate solution was acidified with 20 mi. of cold 6N hydrochloric acid. The acid was added dropwise while the flask was rotated in order to avoid accumulation of excess acid. This precaution eliminated the formation of isomers and polymers (1-4, 6, 8). The hydraeones were extracted from the acidified solution with two 2Bml. portions of ether. The ether extract was dried with anhydrous sodium sulfate and then decanted into a test tube. The sodium sulfate was washed with 2 ml. of ether. The ether extracts were combined and the ether was removed by immersing the tube into ho$ wates. Final traces of solvent were removed m vacuo. The dry 2,4dinitrophenylhydraaones were dissolved in 0.2 to 0.4 ml. of ethyl alcohol. The blood keto acid hydra- zones were dissolved in about 0.1 ml. of ethyl alcohol.

One of the apparatus em loyed in this work was that of Biophysical Instruments, Inc., ghiladelphia, Pa. It operated close to 150 volts and 11 ma. Two determinations were made simultaneously using 285 X 65 mm. Whatman No. 1 filter paper strips. One hundred microliters of hydram solution, equal to 10 ml. of blood, were applied on a line with capillary pipet, 80 mm. from one end of the strip. The 0th instrument employed was that of Grassmsnn and Hannig ( Miltenhere. Iuc.. New Yark. N. Y.). It onerated close to 1

Paper Electrophoresis.

volts and 76 ma.' For this appar&, howekr, i t was necessa to construct a special frame or rack holding one 285 X 80 mi strip. The original frame was devised to hold two narrow stri! The parts for the new frame were cut from Plexiglas and annealed with acetone. As this aDDaratUs Dennits the use of three cham-

ne # a Figure 2. Electrophoretic pattern showing- spots ,er for 2,4-dinitrophenylhydraaones of e-ketoglu tario

acid (ICG), a mixture of hydrazones of pyruvic acid E' a n d a-ketoglutaric acid (PA + ICG), a n d of pyruvic

aoid (PA) 10 ry m. )8:

There x- 4 of each keto aoid6n free acid basin) in Z d. of ethyl +?h*l . .. . .. ~ .. . .

hers, three determinsti&s were n&de simultmeously

V O L U M E 27, NO. 2, F E B R U A R Y 1 9 5 5

(24 nil. or more when other than human blood was analyzed) to the tube containing pyruvic acid hydrazone shreds. The tubes were stoppered with a rubber stopper, shaken for 2 minutes, and centrifuged for 10 minutes. The supernatants were placed in Klett tubes. If filter fibers were present, the tubes were re- centrifuged. The color intensities were measured, without fur- ther decanting, in a Iilett-Sumerson photoelectric colorimeter using green filter No. 51. The keto acid values were obtained from t.he calibration curves (Figure 1).

Separation of Pure Pyruvic Acid and a-Ketoglutaric Acid Hydrazones. Figure 2 shows a filter paper strip where the hy- drazoiie of 4 y of pyruvic acid in 2 pl, of ethyl alcohol was applied to a 285 X 80 mm. strip (uppermost spot). In the second row n mixture of the hydrazones of 4 -/ of pyruvic acid and 4 y of a-ketoglutaric acid was applied; and in the lowest spot the hydrazone of 4 y of a-ketoglutaric acid was applied to the strip and subjected to paper electrophoiwis for 3 hours. It is seen that the mobility of a-ketoglutaric acid hydrazone is greater than that of pyruvic acid hydrazone. The separation of pyruvic acid hydrazone from a-ketoglut,aric acid hydrazone is distinct. The hydrazones were freshly prepared. They moved anode- ward.

In Figure 1 are shown calibration curves obtained when mix- tures (7.5 to 38.0 y) of pyruvic acid and a-ketoglutaric acid were converted t,o their 2,+dinitrophenylhydrazones and subjected to paper electrophoresis. The different concentrations of hydra- zone solutions were obtained by diluting a stock solution of the two hydrazones with ethyl alcohol. The graph shows that the hydrazone of pyruvic acid gives a much more intense color with 1N sodium hydroxide than the hydrazone of a-ketoglutaric acid. Separation of the two hydrazones appears to be quant,ita- tive. There is a proportionality between color intensity and hydrazone concentration in accordance with Beer’s law. Figure 2 was obtained by spraying the strips, after electrophoresis and drying, with 1N sodium hydroxide using a glass sprayer (Re- search Equipment Corp.) attached to a Gast rotary pump. Here separation is didnct . Pyruvic acid hydrazone solutions should not be kept for too many hours a t room temperature; otherwise isomerization and polymerization occurs (1-4, 6, 8).

Freshly drawn blood (1 5 ml.) was added at once to 105 ml. of water and 15 ml. of 0.66N sul- furic acid. This was mixed and 15 ml. of 10% sodium tungstate were added. Aft,er shaking for 1 minute, the mixture was kept for 10 minutes a t room temperature, t,hen centrifuged for 7 min- utes in a refrigerated centrifuge a t 5’ C. If desired, an ordinary centrifuge may be used. The supernatant was filtered through a small folded filter paper. In the recovery experiments the standard was added t,o the whole blood. Human blood was taken from the antecubital vein (laboratory personnel), sheep blood from the jugular vein, and rabbit and guinea pig blood wits obtained by cardiac puncture. All bloods were taken under non- fasting conditions.

To 100 ml. of blood filtratc 5 ml. of 2.4-dinitrophenylhydrazine were added and mixture was treated similar to the pure keto acids. The dry hydrazone residues were dissolved in 80 micro- liters of ethyl alcohol. The teet tube was “rinsed” with 20 pl. of ethyl alcohol and this portion too was applied tjo t8he strip.

Application to Blood Analysis.

This was applied to the strip.

In Table I are recorded pyruvic acid values for rabbit, sheep, guinea pig, and human blood. Neish’s values for rat blood (6) are included for comparison. Rabbit blood contains several times more pyruvic acid than the blood of other niamninls. Differences in a-ketoglutaric acid values (Table 11) are not as marked except for guinea pig blood, which contains very small quantities of a-ketoglutaric acid.

The recovery of added pyruvic acid to rabbit blood (see typical results in Table I) ranges from 96 to l l O % , with a mean of 99%. The recovery of a-ketoglutaric acid (Table 11) ranges from 81 to 85%, with a mean of 83%. When niixtures of pure pyruvic acid and a-ketoglutaric acid were analyzed the recovery was 90 to 95%.

Often a band appears between the pyruvic Component X.

289

Table I. Pyruvic Acid i n Different Mammal ian Bloods ( y per 10 ml. of blood)

Type of Mammal 1 2 3 4 5 6 Mean Rabbit 288 216 216 200 188 296 234.0 Plus 100 y pyruvic acid 376 296 326 288 296 392 . . . Recovery, 70 97 94 110 88 108 96 9 8 . 8 Human Sheep Guinea pig R a t (6)

98 70 71 76 86 74 78 .8 176 188 122 128 110 I50 143.7 RO 128 80 72 90 142 83 .7

118 121 126 D l i 59 79 9 4 . 8

Table 11. a-Ketoglutaric .kcid in Different Mammalian Bloods

( y per 10 ml. of blood) Type of Slaminal 1 2 3 4 5 6 Mean

Rabbit 54 50 52 36 23 51 44 3 Plus 100 y a-ketoglutaric

Recovery. % 85 85 82 82 84 81 83 .2 acid 132 128 118 118 107 132

Hunian Sheep Guinea pigo R a t ( 6 )

21 20 17 25 20 14 19 .5 40 57 38 40 3.1 76 4 7 . 5 8 11 10 6 7 13 9 . 2

48 33 13 22 18 51 30 8 * Here. because of the unusually low a-ketoglutaric acid content, 25 ml.

T h e values, however, are per 10 ml. of blood were employed in the analysis.

wid and the a-ketoglutaric acid hydrazone bands when mamma- lian blood hydrazones are separated on filter paper. This band between pyruvic acid and a-ketoglutaric acid has been observed by Xeish (6) in rat liver extracts but not in extracts of other rat tissue or rat blood. While the present paper was in press, Neish (6), advised this author that he also observed component X in some rat blood filtrates. He has named this band Spot X (6). It was observed in the present work that when the dried strips were sprayed with 1N sodium hydroxide spot X gave a light yellow color as opposed to the usual dark red-brown and dark Ilrown color of pyruvic acid and a-ketogutaric acid hydrazones. Whenever the center band X did not separate, its presence wya9

identified as follows: The a-ketoglutaric acid bar was cut in the middle and immersed in 1N sodium hydroxide; the slower moving half of the bar gave a yellow color, whereas the faster moving a-ketoglutaric acid hydrazone bar gave a dark brown color. Component X is probably not an isomer of pyruvic acid or a- ketoglutaric acid hydrazone. If green filter No. 54 is used, the Iilett reading of this component is negligible. Component X does not appear when pure hydrazones are subjected to paper electro- phoresis.

Neish (6) did not notice this marked difference between the keto acid hydrazones and component X because by using the polarographic method he worked in an acid medium in which all hydrazones were yellow.. He included spot X in the a-ketoglu- taric acid hydrogen spot. The identity of component X is not known at present. 2,4Dinitrophenylhydrazine reagent givep a grayish brown color with I N sodium hydroxide. This reagent, houever, is removed during the purification of the 2,Pdinitro- phenylhydrazones. The bands of pyruvic acid and a-ketoglu- taric acid as separated by the present method may include tracw of other keto acid hydrazones

LITERATURE CITED

(1) Cavallini, D.. and Frontali, N., Biochem. et Biophys. Acta, 13.

(2) Cavallini, D., Frontali, K., and Toschi, G., ,\‘atitre, 164, 792

(3) Isherwood, F. A., and Cruickshank, D. H., Ib id . , 173, 121 (1954) (4) Kulonen, E., Carpen, E., and Ruokolainen, T.. Scand. J . CEin. &

(5) Neish, W, J. P., private communication. (6) Neish, W. J. P., Rec. trav. chim., 72, 105 (1953). (7) Price, 1’. E., and Levintow, I,., Biochem. Preparations, 2, 2 2

(8) Seligson, D., and Shapiro, B., -AKAI.. CHEM., 24, 754 (1952).

RBCEIYED for review July 29, 1954.

439 (1954).

(1949).

Lab.Invest., 4, 189 (1952).

(1952).

Accepted October 29, 1954.