Estimation of Tartronate in Tissues LAURENCE G. WESSON Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass.
b Tartronate in plant and animal tissues i s estimated b y a method in which the dialyzed and partially purified tissue tartronate is converted to glycolic acid b y dilute sulfuric acid. The absorbance of the product formed with Eegriwe's reagent i s compared with that similarly obtained from a known amount of pure tartronic acid. The wide difference between the characteristics from tartronic and glycolic acids between 4800 and 5800 A. makes possible identification and determination of the amount of tartronate in the tissue, and evaluation of the degree of interference b y other components of the dialyzate.
T
significance of tartronate (hydroxymalonate) in metabolism ( 7 ) makes its evaluation in animal and plant tissues desirable. KOmethod for accomplishing this has been reported in the literature. The present procedure is based on the decarboxylation of tartronic acid by dilute sulfuric acid and the spectrophotometric determination, after reaction TI ith Eegriwe's reagent, of the amount of glycolic acid thus formed. HE POSSIBLE
APPARATUS
Spectrophotometer, recording, hpplied Physics Corp., Pasadena, Calif., Cary Model 12M, using a 20-nim. quartz cell, with the slit-n-idth control set at 15 and the dynode set a t 4. p H meter, Beckman Model G with a Xo. 1190-SO general purpose glass electrode. Homogenizer, Potter tissue-crushing, Central Scientific Co., S o . 44148. Pipet, blood, 0.1 ml., Central Gcientific Co., S o . 41002-B. h small bulb was attached, with a screw clamp for accurate adjustment. Dialyzer membrane, scamles cellulose tubing, Central Scientific Co.,
KO.70160. REAGENTS A N D STANDARDS
Calcium hydroxide, blallinckrodt, analytical grade. Eegriwe's reagent (3), prepared by dissolving 20 mg. of 2,T-dihydroxynaphthalene (Eastnian Kodak Co., Rochester. N. Y., Catalog S o . 4408) in 100 nil. of concentrated, C.P. sulfuric acid, allowing the greenish yellon- solution to decolorize itself a t room temperature and thereafter keeping it chilled. The yiolet color reaction was used by Eegrin-e for the detection of glycolic 1080
ANALYTICAL CHEMISTRY
acid (S), and by Rieben and Hastings (5) for the detection of tartronic acid after pyrolysis to glycolic acid. Lead acetate, Pb(C2H30&.3Hz0,piepared (6) from Baker's reagent grade lead monoxide and glacial acetic acid [Mallinckrodt, analytical grade, purified b y potassium permanganate treatment ( S ) ] , and used as a solution saturated a t room temperature in dilute acid (1 volume of purified glacial acetic acid to 4 volumes of water). Potassium carbonate, Mallinckrodt, analytical reagent, in 1.5M solution. Sodium hydroxide, pellets, Baker, analyzed, in 3.V solution. Sulfuric acid, C.P. reagent, concentrated, in 3 5 solution. Tartronate standard solutions, from 5, 10, 15,and 20 mg. of tartronic acid prepared by Bak's (1) method; decomposition point, 155' C. with 10" rise per minute (156"to 158" C.according to Bak); equivalent weight, 60.39 grams (theoretical, 60.03 grams), and 1 ml. 3.V sodium hydroxide made to 50 ml. These solutions retain their tartronate value well (0.1 ml. 0.01, 0.02, 0.03, and 0.04 mg. of tartronic acid). Tartronic acid of suitable quality is manufactured b y the Aldrich Chemical Co., Milwaukee, Wis. Glycolate standard solution, from 6.2 mg. of crystalline glycolic acid (melting point 76-So C., Eastman's Catalog No. 998) and 5 ml. of 3 N sodium hydroxide, made to 50 ml. (0.1 ml. == 0.02 mg. of tartronic acid). PROCEDURE
The weighed ground or thin-sliced sample (1 to 10 grams, depending on the result obtained in a preliminary test) was acetone-extracted. After expulsion in vacuo of the acetone remaining in the sample, it was moistened with water and hard-frozen, melted, and homogenized (ice-water bath). The prepared sample was dialyzed into 1 liter of water (ice box) for 3 days and into a second liter for two additional days (to test the completeness of the dialysis). I n the meantime the acetone extracts were combined and filtered, the acetone was evaporated a t room temperature, and the lipides in the iesidue nere hydrolyzed by stirring the residue with a n excess of a thin slurry of about 0.15 gram of calcium per gram hydroxide of the oil (4). The amount was roughly gaged from tables giving the composition of tissues or of foods similar to the sample. Heat was applied by means of a steam bath for 15 minutes, after which the mixture stood overnight (4). The insoluble soaps and unsaponifiable
matter were centrifuged off and washed well with water, and the supernatants combined and filtered. The filtrate, containing the sufficiently soluble calcium tartronate (equivalent to about 0.1 mg. of tartronic acid per ml. of saturated calcium hydroxide solution a t 25" C.), was added to the dialyzates. These were evaporated to dryness after adding potassium carbonate, if necessary, to at least p H 8. T o precipitate the tartronic acid, the residue was taken up with sufficient dilute acetic acid t o give p H 4.5, filtered, and the filtrate adjusted to p H 5.5 with sodium hydroxide solution. Saturated lead acetate solution n a s added drop by drop until the precipitation was complete a t that pH, as shown by repeated testing and centrifuging. The volume of the supernatsnt wis measured a t this point to allow calcul Ition of the weight of the tartronic acid in solution. To eliminate the leid, the precipitate was stirred intermittently with 1 ml. of 3.V sulfuric acid for half an hour, centrifuged out, and washed several times. The combined supernatants were made p H 5 0 with sodium hydroxide solution, and the small residual amount of h a d precipitated as sulfide and filtered off into a 5or 10-ml. volumetric flask. T h r solution was freed from hydrogen sulfide by a stream of small bubbles of filtered air, alkalized to litmus paper, and made to volume. After again filtering the solution, a n aliquot (0.1, 0.2, or 0.3 ml.) was pipetted into a soft-glass test tube and evaporated to dryness by means of a boiling water bath and a stream of filtered air. An aliquot (0.1, 0.2, or 0.3 ml.) of the standard tartronate was likewise pipetted into a second sbft-glass test tube and evaporated. The tartronate in the two test tubes was decarboxylated to glycolic acid HzSOP(HOOC*CHOH.COOH7 HOOC * CH20H C02) as follows: One milliliter of 3iv sulfuric acid was added to each tube and the carbon dioxide stemming from the alkali was expelled by a current of filtered air. The two test tubes were then sealed and heated (suspended side by side) for 15 or 16 hours in an oven a t 100" C. After the contents of each had been cooled and transferred to hard-glass test tubes n i t h 1 ml. of 1.5X potassium carbonate and wash water, they were evaporated as before until thoroughly dry. The solutions and the absorbances eventually derived therefrom are designated i i D (acid and dialyzate) and AS (acid and standard). T o a third tube was added dialyzate as for .4D and to a fourth, standard
+
12
o c
I 4800
I
~
5800 4800
I
I
I
I
I
4800 5800
5800
Angstroms
Figure 2. Absorbance characteristics D ) and (AS S) made with for (AD double beam
-
0
4800
1 5800 4800
5800
Angltromr
Figure 1. Absorbance characteristics for AD, D, AS, and S made with sample beam a , b.
Purified dialyzate of liver, with and without decarboxylation c, d. 0.02 mg. of tartronic acid, with and without decarboxylation
tartronate as for A S . These solutions were evaporated t o dryness together with their eventual absorbance, and are called D and S. The color was developed by adding 5 ml. of chilled Eegriwe's reagent to each of the four tubes and heating these for 30 minutes, suspended side by side, in a boiling water bath. The solutions were cooled and rinsed into 10-ml. volumetric flasks with concentrated sulfuric acid to volume. Absorbance characteristics between 5000 and 5600 -4.(or if desired as a further check, 4800 and 5800 A.) were recorded within a n hour, using the sample beam alone (Figure 1) or the double beam (Figure 2 ) to obtain (-4D - D ) and ( A S - 8). CALCULATIONS
Assume that no component other than tartronic acid is altered by sulfuric acid or b y Eegriwe's reagent in such a way that the ratio of increased absorbance ( A D - D ) to increased absorbance (AS - S) is appreciably affected betiyeen 4800 and 5800 A. The weight of tlie glycolic acid formed from the tartronate of the dialyzate and that from tlie standard tartronate will therefore be proportional, if suitably corrected, to ( A D - D ) / ( A S - 8). Thus, ( A D - D cd)/(AS - S c,) = p2x/p,s, where 2 is the weight of tartronic acid in the sample of dialyzate taken for analysis; s, that of the standard tartronic acid; p,, the percentage con-
+
+
a , b. c.
-
Parallel dialyzates of apple 0.04 mg. of tartronic acid
version in A D ; p , , that in A S ; and cd and c8 are the net corrections for the dialyzate and standard, respectively. The percentage conversion will, under the parallel conditions, be the same for A D as for A S and cancels out. Thus 5 = s(AD - D cd)/(AS - S c~). To evaluate c d and e, two effects must be heeded: 1. A D and A S values require a n appreciable but equal correction CK2C03 because of the potassium carbonate used just prior to Eegrin-e's reagent. T o determine the magnitude of this, 1 ml. of 3N sulfuric acid was neutralized b y 1 ml. of 1.5M potassium carbonate, the solution evaporated to dryness, and the residue heated 30 minutes with 5 nil. of Eegriwe's reagent. The blank was 5 ml. of reagent heated in parallel and made to 10 ml. with concentrated sulfuric acid. Readings of the increased absorbance due to the potassium carbonate were made in the n-are-length
+
+
range of interest (4800 to 5800 A.) and ~S comprised the negative C K ~ C O ~ that were applicd to the ( A D - D) and (AS - S) readings a t tbe same wave lengths. 2. The D and S values require a smaller and positive correction C T 4 because of the tartronic acid (TA) that disappears in the form of glycolic acid and so should not be included in the base-line values. It is shown in a later paragraph that the conversion may be considered approximately complete under the conditions of the procedure. Because the effect of the tartronic acid-Eegrin e's reagent product on absorbance is very small (Figure 3), the CTL found for S may be used also for D . The C T values ~ Lvere found for each weight of the standard (0.001, 0.02, 0.03, and 0.04 mg.) by measuring the effect produced a t the wave lengths of interest by these amounts of tartronic acid, the blank, as for CK2C03. being the reagent in each case. Because c d and c, are approximately the same, they may be replaced by c; c then is the algebraic sum of CK2CO3 (which is larger than CT.4. negative, and dependent on the ware length) and of C T 4 (which is positive and dependent on both wave length and s). The use of c is unnecessary if (AD - D) E (AS
- S).
I n Table I the measured and calculated results are given in detail for one tissue. using 1-gram samples. The purified dialyzates were made to 10 ml., 0.1-nil. aliquots were used, and 0.04 mg. of tartronic acid was the standard. Absorbance characteristics are shown in Figures 1 and 2. The essential calculated data obtained are listed for a number of plant and animal tissues in Table
11. TEST OF THE ASSUMPTION
If the corrected ( A D - D) values represent solely the formation of glycolic from tartronic acid, the z's calculated for all wave lengths in the range for which the increased absorbance is
Table 1. Duplicate Determinations of Tartronic Acid Content of Apple
Kave Length A. 5000 5200 5400
5600
5000
5200
Absorbance (AD - D ) 0 981 1 178 1 399 1 145
1 0T9 1 257
(AD
-D
- c)
0 902 1 106
1 330 1 097
(AS - S ) 1 290 1 474
1 573
-s
(AS - c)
1 211 1 402
1 501
1 3i5
1 327
1 000
2,
Mg. 0 0298 0 0316 0 0353 0 0331 Av. 0 0325
1 211
1 185
Tartronic acid per gram of apple: Av. x,,,,. = 0.0338 - (0.0400 - 0.0338)/10 = 0.0332 mg. Wt. TA in sample = 0.0332 X 10/1 X 0.1 = 3.32 mg. Corrected for solubility of lead tartronate in 5.6-ml. solution, this becomes 3.32 5.6 X 0.010 = 3.38 mg. of TA per gram of apple.
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VOL. 30, N O . 6, JUNE 1958
1081
Table II. Tartronic Acid Content of Diverse Plant and Animal Tissues" T.4 per Purified TA in Gram Wt. per Dialyzate TA in Corr. Av.b Wet Sample, Nade to Aliquot, Std., Aliquot, S.D., Sample, Grams ml. ml. Mg. Mg. Mg. % Celery 2 0.14 6 0.02 0.0133 0.3 4.9 0.15 0.0138 Lettuce 0.0347 3 10 0.3 1.4 0.41 0.04 0.40 0.0344 1 3.23 0.04 0.0317 10 0.1 3.7 Apple 3.38 0.0332 Potato 2 0.1 0.0210 10 0.38 0.02 0.3 0.0182 0.34 Walnut 2 0.02 0,0145 0.2 10 0.39 3.2 0.0116 0.33 0.01 Heart muscle (veal) 0.0146 2 0.15 0.01 5 4.6 0.3 0.0267 4 0.12 0.02 Heart (acetone-extracted) 1 0.0143 0.55 0.02 10 3.6 0.3 0.0125 0.49c 0.0161 Kidney (veal) 0 0.02 4.7 0.3 10 0.04 0.0152 0.04 Liver (veal) 0.0205 2.5 0.3 5 5 0.02 0.08 0.0189 0.08 Adipose (beef) 5 0,0083 i) 0.01 0.04 4.3 0.3 0.0083 0.04 a Duplicate complete analyses (including dialyses) were made, using two samples from same mixed-ground or sliced portion. Tartronic acid content varies somewhat from one specimen to another, and according to variety, season, age, and so forth. To make certain that absorbance represented by each characteristic is predominantly that of glycolic acid formed by acid-heat treatment, standard deviation [ ( Z d 2 ) / ( n- 1)]1'* of four 5 ' s was calculated for each of parallel analyses from readings at 5000, 5200, 5400, and 5600 A. Two standard deviations Tere then averaged. c Based on dry weight. Approximation previously given ( 7 ) has been found to be incorrect.
sufficiently great should be the same because a simultaneous conversion from pure tartronic acid is used in deriving the (AS - S) curve. Between 4800 and 5800 A. the absorbance component produced b y glycolic acid undergoes a n extremely sharp and considerable rise to and fall from its maximum a t about 5400 A. (Figure 3, a and c), while a lorn, almost straight and only slightly sloping characteristic is given by tartronic acid ( b and d). A coincidental agreement of 5 over a range of this character would be extremely unlikely. A small deviation from the mean indicates that little or no interfering substance is present. A large percentage deviation-eg., 10~o-from the mean indicates too great an uncertainty caused by a n interfering substance or substances in the purified dialyzate. Further washing of the lead acetate precipitate or a second precipitation may be required to improve the adherence of ( A D D ) to its corresponding ( A S - S) curve. DISCUSSION
Possible Decarboxylation during Dialysis. To ascertain whether t h e acidity ( p H 4 t o 6) of t h e dialyzate resulted in t h e conversion of p a r t of 1082
ANALYTICAL CHEMISTRY
t h e tartronic t o glycolic acid during dialysis, 4 mg. of tartronic acid were added t o 1 liter of water. T h e solution was made p H 3.5 and allowed t o remain in t h e ice box for 3 days. It was then alkalized, evaporated t o a small bulk, and made u p t o 10 ml. When tested in parallel with 0.04 mg. of standard tartronic acid, no difference between the two absorbance characteristics was apparent and thus no detectable decarboxylation occurred. Solubility of Lead Tartronate. T h e solubility of lead tartronate in water at room temperature and p H 5.5 in t h e presence of a n excess of lead acetate was found to be t h e equivalent of 0.010 mg. of tartronic acid per ml. This value multiplied b y the volume of t h e supernatant from t h e lead precipitate gave a correction which was added t o t h e weight of tartronic acid in the analyzed sample (Table I). More tartronate could be recovered by using a less acid solution, but the possibility of precipitating interfering substances led to the choice of the lower PH. Purification of Dialyzate. This difficult solubility of lead tartronate in acid solution made possible the separation t o a considerable degree of tartronate from other components,
O
M [
'
'
!
I
4800
I
O
5800
I
I
I
I
' 5
4800
Angstroms
Angstroms
Figure 3. Ab- Figure 4. Absorbsorbance charac- ance characteristeristics tics b. 0.02 mg. of tartronic acid, with and without decarboxylation c, d. 0.04 mg. of tartronic acid, with and without decarboxylation a,
2.5 mg. of apple, without purification or decarboxylation b. 10 mg. of apple, with purification but without decarboxylation c. 10 rng. of apple, with purification and decarboxylation
a.
especially from most of t h e large glycolate content (Figure 4, a). Although t h e dialyzate was still far from pure (Figure 4, b ) , t h e absorbance given b y t h e newly formed glycolate could be made nearly equal to that from a feasible tartronate standard (Figures 1 and 2). hloreover, the small standard deviation of the x's obtained in the optical range of interest showed (Tables I and 11) that it was relatively free from interfering substances; the effect of the remaining impurities was the same for both A D and D,and ( A D - D ) was therefore relatively unaffected (Figure 1). The purification method was also tested as to loss of tartronate. Standard tartronate (10 ml., containing 2 mg. of tartronic acid) was evaporated in a graduated centrifuge tube to about 5 ml. and processed as in the procedure. The total obtained was 1.98 mg. of tartronic acid, including 0.054 mg. in the 5.4 ml. of supernatant from the lead acetate precipitation. The removal of the lead present in this lead acetate precipitate was necessary because of a n adverse effect of the lead on the Eegriwe color reaction. Removal by direct action of hydrogen sulfide was not feasible because of the inclusion of part of the difficultly solu-
ble tartronate in the still less soluble sulfide. However, b y dissolving the lead tartronate in a more strongly acid solution and, a t the same time, precipitating most of the lead as sulfate, it was possible thereafter to return the p H to a value a t which the small remaining amount of lead could be converted to sulfide with no tartronate included. A test was made as to tlie possible decarboxylation of the tartronic acid in this half hour’s treatment n i t h 3*1’sulfuric acid a t room temperature. Using the procedure with a qolutioii containing 2 mg. of tartronic acid, it was found that the resultant D characteristic n as unchanged from that obtained n ith the same aniount of 1111treated tartronate; thus no appreciable decarbo\r.lation occurs. Decarboxylation of Tartronic Acid.
I n connertion n i t h the choice of time, temperature. acid concentration, a n d espwially with t h e calculation of C T ~ . t h e appi o u n i a t e percentage conveision of tartionic t o glycolic acid Ti-as determined. Glycolate equivalent t o 0.04 nig. of taitronic acid v a s heated with Cegrin e’s reagent in parallel rvith this ieagent containing no glycolate. This simulated 100% conrersion of 0.04 nig. of tartronic to glycolic acid and gave an incrpased absorbance a t 5400 A. of 1.45. Thp percentage conversion of 0.04 nig. of tartronic acid was calculatcd as the increased absorbance found by the decarboxylation procedure dirided by 1.45. The heating
period finally selected for the procedure was 15 to 16 hours, because the increased absorbance (corrected) a t 5400 A,, using 0.04 mg. of tartronic acid, was only 1.05 at 6 hours; this corresponded to about 7OY0 conversion, and 1.37, giving 947, at 10 hours, while a t 15 to 16 hours it Tvas 1.41, corresponding to about 97% conversion.
acid (in p H 7 solution) prior t o dialysis; none was added t o t h e other sample. T h e analysis for t h e second sample gave 0.46 mg. of tartronic acid per gram of potato and for the first, 0.69 mg., instead of 0.66 mg. This indicates complete recovery of the added tartronic acid.
Proportionality of Increased Absorbance for Tartronic + Glycolic Acid. This ratio includes not only
ACKNOWLEDGMENT
adherence t o Beer’s law and rate of formation of t h e colored compound produced by Eegriwe’s reagent from glycolic acid, but also the rate and percentage conversion of tartronic to glycolic acid b y dilute sulfuric acid. Tartronic acid samples, two of 0.02 mg. and tiyo of 0.04 mg., ITere evaporated. One of each was heated with sulfuric acid. The result: 0.02 mg. gave an increased absorbance (corrected) a t 5400 d. of 0.784, while tlie 0.04-mg. sample gave 1.454, or about 90% of 2 X 0.234 (Figure 3). T o compensate for this lack of direct proportionality, the difference betn een the u-eight of the standard used and the average z of the corresponding aliquot was divided by ten and the quotient subtracted from (if the standard was tlip larger) or added to the average z. This x-alue was used as tlie average zcorr(Table I). Recovery
of
Added
The author is grateful to A. R. von Hippel, professor of electrophysics and director of the Laboratory for Insulation Research, Massachusetts Institute of Technology, for the use of the spectrophotometer , LITERATURE CITED
(1) Bak, B., ilnn. 537,286-92 (19391. (2) Bousefield, If-. K., Lon-ry, T. AI., J . Chem. SOC.(London) 99, 1432-41 (1911). (3) Eegriwe, E., 2. a n d . Cheni. 89, 121-5 (1932): (4) Lewkowitsch, J., ‘.Chemical Technology and Analysis of Oils, Fats and Waxes,” p. 208, Vol. 3, Nacmillan, London, 1915. ( 5 ) Rieben, IT. K., Hastings, -4. B., H e l ~ .Physzol. et Pharnzacd. &fa 4, (252-3 (1946). (6) Tliorpe, J. F., Khiteley, hI. A., “Dictionary of Applied Chemistry,” 4th ed., 5’01. 1, p. 55, Longmans, Green & Co., Sew Tork, 193T. (Tj Kesson, L. G., Science 122, 595-7 (1955).
Tartronate.
T o one duplicate 2-gram sample of potato \vas added 0.40 mg. of taitronic
RECEIVED for review December 10, 1956. -4ccepted Januar? 22, 1958.
Mass Isotope Dilution Assay for Gibberellic Acid B. H.
ARISON, 0. C. SPETH, and
N. R.
TRENNER
Merck Sharp & Dohme Research laboratories, Division o f Merck & Co., Inc., Rahway, N. J.
,An isotope dilution assay particularly suitable for fermentation broths has been developed for gibberellic acid. The tracer i s prepared b y treating gibberellic acid with 85% deuterioacetic acid at 56” C. in the presence of a platinum catalyst. The analytical procedure involves addition of a known weight of tracer to a system containing unlabeled gibberellic acid, qualitative isolation of a pure isotopically mixed specimen, combustion of the isolate, and infrared determination of the per cent deuterium in the water so produced. The accuracy is to about &4y0.
I
to the increasing agricultural interest in gibberellic acid. efforts a t the llerck Laboratories have been directed toward improving proi~ RE‘POXSE
duction methods for this gron-th stiniulant. Essential was a reliable method of analysis that would allom- unequivocal decisions on the relative merits of various processes. Several widely divergent approaches were employed, including biological, chemical, fluorescent, and paper strip ( 4 ) . The fluorescent technique, which depends on color development in sulfuric acid, is simple and rapid. although its reliability, particularly in fermentation broths, is not aln-ays unchallengeable. This limitation also holds for the other procedures. Such erratic performance is not surprising, as complex media often contain substances which influence the analytical response. However, an analytical method was required nhich was unaffected by the nature of the medium. This requirement is satisfied b y the isotope dilution technique, which demands onlv the
qualitative isolation of s pure specimen of the isotopically mixed analogs to be specific-Le., accurate. Based upon previous succeSs with analytical problems of the same general type (2, 7 , 8). deuteriuni was selected as the tracing isotope. It was introduced into the gibberellic acid molecule b y a technique similar to that of Fukushima and Gallagher ( I ) , in which platinum catalyst and 85% deuterioacetic acid were used. -4knoivn quantity of deuteriogibberellic acid was added to a fermentation broth aliquot, and a pure specimen of the mixed labeled and unlabeled materials was qualitatively isolated. The atom per cent deuterium in this isolated product is determined by infrared analysis of the water of combustion according to the method of Trenner, Arison, and Kalker (6). From the dilution of the deuterium, the VOL. 30, NO. 6, JUNE 1958
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