GASOMETRIC DETERMINATION OF OXALIC ACID AND CALCIUM, AND ITS APPLICATION TO SERUM ANALYSIS.* BY DONALD D. VAN SLYKE AND JULIUS SENDROY, JR (From the Hospital of The Rockefeller Institute for &fedical Research, New York.) (Received for publication, July 12, 1929.) The reaction between oxalic acid and permanganate indicated by the equation 5HG04 + 2KMnOd + 3H&04 = K2SOa + 2MnSOc +8H,O +lOCOg is the basis of most of the calcium methods current in biological chemistry, the amount of oxalate in precipitated calcium oxalate being determined by titrimetric measurement of the permanganate utilized. In this paper we present results showing that the oxalate can be determined with equal accuracy by measurement in the Van Slyke-Neil1 (1924) manometric apparatus, and that the gaso- metric procedure can be applied with especial advantage to micro determination of serum calcium. The advantage of the gasometric method in micro analyses is indicated by the fact that the oxalate precipitated by 0.1 mg. of Ca, the amount in 1 cc. of ordinary serum, reduces in titration only 0.50 cc. of the 0.01 N permanganate used in the Kramer and Tisdall (1921) method, while by the gasometric procedure the COZ produced exerts at 0.5 cc. a pressure of about 160 mm. to read on the Van Slyke-Neil1 manometer. The permanganate without unusual precautions can hardly be measured with better than 4 per cent accuracy, while 1 per cent is easily attainable in the manometric measurement. With larger amounts of oxalate to determine, titration and gasometric measurement yield precise and identical results. * A preliminary note on the method appeared 3 years ago (Van Slyke and Pcxndroy, 1926). 217 by guest on November 14, 2018 http://www.jbc.org/ Downloaded from
Transcript
GASOMETRIC DETERMINATION OF OXALIC ACID AND CALCIUM, AND ITS APPLICATION TO SERUM
ANALYSIS.*
BY DONALD D. VAN SLYKE AND JULIUS SENDROY, JR
(From the Hospital of The Rockefeller Institute for &fedical Research, New York.)
(Received for publication, July 12, 1929.)
The reaction between oxalic acid and permanganate indicated by the equation
is the basis of most of the calcium methods current in biological chemistry, the amount of oxalate in precipitated calcium oxalate being determined by titrimetric measurement of the permanganate utilized. In this paper we present results showing that the oxalate can be determined with equal accuracy by measurement in the Van Slyke-Neil1 (1924) manometric apparatus, and that the gaso- metric procedure can be applied with especial advantage to micro determination of serum calcium.
The advantage of the gasometric method in micro analyses is indicated by the fact that the oxalate precipitated by 0.1 mg. of Ca, the amount in 1 cc. of ordinary serum, reduces in titration only 0.50 cc. of the 0.01 N permanganate used in the Kramer and Tisdall (1921) method, while by the gasometric procedure the COZ produced exerts at 0.5 cc. a pressure of about 160 mm. to read on the Van Slyke-Neil1 manometer. The permanganate without unusual precautions can hardly be measured with better than 4 per cent accuracy, while 1 per cent is easily attainable in the manometric measurement. With larger amounts of oxalate to determine, titration and gasometric measurement yield precise and identical results.
* A preliminary note on the method appeared 3 years ago (Van Slyke and Pcxndroy, 1926).
Because of the difficulty of obtaining precise results with the permanganate titration in micro analyses Hamilton (1925) and Trevan and Bainbridge (1926) have introduced an extra step. They heat the calcium oxalate precipitate until it is changed to calcium oxide (Hamilton) or carbonate (Trevan and Bainbridge), dissolve the residue in excess hydrochloric or phosphoric acid, and titrate back with alkali. The procedure is less simple than the direct permanganate titration, and presumably for that reason the latter appears to continue in general use.
The demonstration of a quantitative yield of COZ from oxalic acid treated with excess permanganate in the manometric appara- tus offered no difficulties. The technique for obtaining, pure and without loss, the minute amounts of calcium oxalate crystals which the delicacy of the manometric method permits one to determine has absorbed the greater part of the time required for the work here presented.
The preliminary treatment of blood serum or plasma proved to be a point of importance. Halverson and Bergeim (1917), of whose blood calcium method most subsequent ones are more or less successful modifications, precipitated the proteins with picric and hydrochloric acids, and brought the filtrate to the proper slightly acid reaction by adding sodium acetate and ammonia. Rothwell (1927) has followed a similar procedure. Other authors, however, (Clark, 1921; Kramer and Tisdall, 1921; Clark and Collip, 1925; Hamilton, 1925; Trevan and Bainbridge, 1926) have precipitated the calcium in the whole serum slightly diluted. In our preliminary note (1926) we also followed this procedure.
Results reported below, however, (see Table IV) indicate that the serum proteins inhibit measurably the precipitation of calcium oxalate. By precipitation in diluted serum we have obtained results 5 to 15 per cent lower than those obtained by precipitation in the protein-free trichloroacetic acid filtrate. It was further noted that after the calcium had been precipitated as completely as possible by ammonium oxalate in diluted serum, when the super- natant mother liquor was deproteinized with trichloroacetic acid and the filtrate brought to pH 5 an additional precipitate of cal- cium oxa1at.e appeared. Quantitatively it made up the 5 to 15 per cent deficit noted above. Accordingly, in our method we pre-
cipitate the calcium in the trichloroacetic acid filtrate rather than in diluted serum.
Another point of technique, and the one that has drawn most attention from previous authors, has been the washing of the calcium oxalate precipitate. As pointed out by Clark (1921) the solubility of calcium oxalate, though slight (7 mg. per liter at 25”), is sufficient to cause significant losses when only 0.2 to 0.4 mg. of precipitate is washed. 10 cc. of water, if in contact long enough to become saturated (which it is not, of course, in any ordinary washing technique), could dissolve 0.07 mg. of the precipitate. When the washing is done by the centrifugation method, there is also opportunity for loss of some crystals in the decanted washings. The losses that can readily occur are exemplified in Table III. Obviously a minimum of washing is desirable. Yet it must be sufficient to remove the ammonium oxalate completely, ‘or so nearly so that the trace left will just compensate for the oxalate lost. The washing procedure empirically found to meet this requirement, will, as stated by Clark and Collip (1925) be “arbi- trary and must be closely adhered to.” Kramer and Tisdall wash by centrifugation three times with 4 cc. portions of dilute am- monia, syphoning off the washings; Clark and Collip wash only once with 3 cc., but obtain more complete removal of mother liquor and washings by permitting the inverted tube to drain for 5 minutes. After numerous trials of similar procedures we finally adopted two washings with 3 cc. portions of dilute ammonia, poured upon the precipitate but not stirred up with it. This technique gave in our hands the most consistent results (e.g. see Table V). However, we consider that the washing process re- mains the part of the analysis to which its maximum error (+3 per cent) is chiefly due. Any errors beyond the above limit readily occur if the washing procedure is not carried out scrupu- lously as directed.
Reagents,
Trichloroacetic Acid, 20 Per Cent.-20 gm. dissolved and diluted to 100 cc., freshly prepared for use.
Sodium Acetate, 20 Per Cent.-20 gm. dissolved and diluted to 100 cc.
Brom-Cresol Green, 0.016 Per Cent.-Prepared from stock solu- tions as described by Sendroy and Hastings (1929).
1 :I Ammonia Water.-Concentrated ammonium hydroxide diluted to twice its volume.
Ammonium Oxalate.-Saturated aqueous solution, about 3.5 per cent.
Ammonium Hydroxide, 2 Per Cent.-2 cc. of concentrated am- monium hydroxide diluted to 100 cc.
Approximately 1 N Sulfuric Acid.-27 cc. of concentrated H&SO* diluted to 1 liter.
Approximately 0.15 N Potassium Permanganate.-4.8 gm. of KMn04 are dissolved and diluted to 1 liter. A portion is acidified before use by the addition of 0.05 volume of 1 N H&Sod.
Approximately 5 N Sodium Hydroxide.-200 gm. of NaOH dissolved and diluted to 1 liter. The solution is conveniently used from a tube with a pinch-cock at the bottom, described by Van Slyke and Neil1 (1924, p. 535) without oil.
All reagents, water, and filter paper should be tested for calcium as an impurity.
Procedure.
Deproteinization.-When there is sufficient material 2 or more cc. of serum or plasma are placed in a measuring flask calibrated to hold 5-fold the volume of the sample. The latter is diluted with about 3 volumes of water, and then 1 volume of a freshly prepared 20 per cent trichloroacetic acid solution is added drop by drop. Water is added up to the mark; the material is mixed and allowed to stand $ hour for precipitation to finish. The mixture is then transferred to a tube and centrifuged. The supernatant liquid is poured off through a small ashless filter paper, and a filtrate is obtained of about four-fifths of the mixture volume.
When only 1 cc. of serum is available, it is precipitated in a 10 cc. flask with 2 cc. of 20 per cent trichloroacetic acid, and as much of the filtrate as possible used for analysis.
Precipitation of Calcium Oxalate in the Filtrate.-To a measured portion of 5 or 10 cc. of filtrate in a scrupulously clean’ 15 cc. graduated centrifuge tube 1 cc. of 20 per cent sodium acetate,
1 When the tubes are not in use we keep them immersed in cleaning fluid (1 gm. potassium dichromate per 100 cc. of concentrated sulfuric acid).
6 to 8 drops of 0.016 per cent brom-cresol green indicator, and 1 cc. of saturated ammonium oxalate are added. In adding the oxalate care is taken that it drops directly into the solution and does not touch the lip of the tube, from which removal by the sub- sequent washing is likely to be incomplete. The mixture is stirred with a thin footed glass rod. A few drops of 1: 1 ammonia are added until the resulting color matches that of a similar volume of a phosphate buffer solution of pH 5.0, containing the same number of drops of indicator. Shohl (1922) has shown that at pH above 4.0 calcium oxalate is completely precipitated. The stirring rod is washed off with a few drops of water. The tube is covered, and the mixture is allowed to stand overnight to complete precipitation.
After precipitation the solution is rapidly centrifuged, and the supernatant fluid is slowly and carefully sucked off without dis- turbing the precipitate. For this purpose we employ the familiar device of an upturned capillary. A piece of thin glass tubing 2 mm. in diameter is drawn out to a capillary with a U bend at the end. The tip of ‘the suction tube is kept below the surface of the solution in the centrifuge tube, to minimize the chance of drawing in crystals still on the surface of the liquid. Solution is with- drawn until only 0.2 or 0.3 cc. is left in the centrifuge tubes. The tube is then washed twice by centrifugation with 3 CC. portions of the 2 per cent ammonia water. In each washing the ammonia is poured gently down the walls so that the latter are washed about their entire circumference with least possible disturbance of the precipitate. The mixture is then centrifuged and the liquid is drawn off as outlined above.
Resolution of Precipitate and Transfer to Chamber of Manometric Apparatus.-2 cc. of 1 N sulfuric acid are run down the wall of the centrifuge tube in such a manner that every portion of the wall is washed. The tube is dipped into hot water to accelerate resolu- tion of the crystals, then cooled to room temperature.
The outside rim of the centrifuge tube is smeared with a thin film of Vaseline, to prevent the solution from creeping over the rim when decanted. The tube is then emptied smoothly, without splashing, into the cup of the Van Slyke-Neil1 chamber, and the solution is drawn down into the chamber. 4 cc. of water are then used, in three portions, to wash the walls of the centrifuge
tube. With the original mother liquors plus a little of the first washing extending down into the chamber to its 2 cc. mark, the washings are collected in the cup above until they reach the 4 cc. mark on the cup. The washings are then run down into the chamber, making 6 cc. of total solution in it.
Liberation and Measurement of CO2 in the Gas Apparatus.-The dissolved air and trace of COZ in the solution are extracted by evacuating the chamber and shaking for 1 or 2 minutes. The extracted gases are ejected according to the technique described by Van Slyke (1927, p. 240), any liquid reaching the cup being allowed to flow back into the chamber. The wall of the cup is washed down with 1 cc. of acidified 0.15 N KMnO,, which is then allowed to run into the chamber. The chamber is evacuated, and, with the mercury at the 50 cc. mark, is shaken for 3 minutes. In this time the oxalic acid is oxidized to COZ and the latter is ex- tracted from solution. The precipitat.e of partly reduced man- ganese oxide which first forms may ent,irely disappear during the last minute, leaving a water-clear solution because of the reducing effect of the mercury in the chamber.
After the COZ is extracted the mercury is allowed to ascend in the chamber, with the precautions outlined on p. 533 of Van Slyke and Neill’s (1924) paper under “Adjustment of Gas Volume” in COZ determinations. If the sample represents 1 cc. or less of serum (0.1 mg. or less of Ca), the gas volume is brought to 0.5 cc. for the pl reading. If the sample represents 2 cc. or more of serum (or over 0.2 mg. of Ca), it is preferable to read pl with the gas at 2.0 cc. volume.
After the pl reading is recorded the cock leading to the leveling bulb is opened, and the bulb is placed at a level slightly below the gas chamber, so that gas in the latter is under slight negative pressure. 1 cc. of 5 N sodium hydroxide, followed by a little mercury, is then admitted to the chamber to absorb the COZ. The pz reading is finally taken with the same gas volume in the chamber as at the pl reading.
Determination of c Correction.-A blank analysis is performed in which 2 cc. of 1 N sulfuric acid and 4 cc. of water are placed in the Van Slyke-Neil1 chamber and analyzed as described above. The PI - p2 difference obtained is the c correction for COZ from the
reagents. The c value is ordinarily about 5 mm. when the gas is measured at 0.5 cc. volume, 1 or 2 mm. at 2 cc. volume.
Calculation.-The pressure Pco, due to COz from oxalic acid is calculated as
Pco, = p, - pz - c
The Pco2 value thus obtained is multiplied by the proper factor in Table I to estimate calcium. Table I has been computed from the CO% factors of Van Slyke and Sendroy (1927), on the assump- tion that each molecule of oxalic acid yields 99.4 per cent of 2 mole- cules of COz under the conditions of analysis, as indicated by the experiment,al work below.
Cleaning Chamber of Manonzetric Apparatus.-After each analysis the apparatus is cleaned, in the manner described on p. 534 of Van Slyke and Neill’s (1924) paper, except that no lactic acid is used. The chamber is washed first with water, then with 1 N
sulfuric acid. It is important for this analysis that the chamber be clean and free of organic matter. Lactic acid is oxidized to COz by permanganate; hence it is essential that no traces of it shall be present,.
EXPERIMENTAL.
Standadization of Oxalic Acid Solutions.
Standard solutions of oxalic acid were prepared and checked by the following methods of analysis.
(a) Titration with sodium hydroxide standardized against 0.1 r‘~ hydrochloric acid made from Hulett and Bonner’s (1909) con- stant boiling HCl, the accuracy of which was checked by gravi- metric silver chloride analyses. The indicator used was phenol- phtbalein.
(b) Precipitation as calcium oxalate in a solution acidified with acetic acid. The precipitated calcium oxalate was washed and then ignited to CaO and weighed as such. This is the standard gravimetric method.
(c) Gravimetric determination of the COz formed by oxidizing the oxalic acid with permanganate. The apparatus previously described by the authors (1927) for the gravimetric determination of COz was used. The oxalic acid with HzS04 was put into the
reaction flask and heated, while an excess of permanganate solu- tion was allowed to drip from the funnel. The COz evolved was collected and weighed in soda-lime U-tubes, as described (Van Slyke and Sendroy, 1927).
In solutions of different lots of acid closely agreeing results by all three methods indicated oxalic acid contents varying from 98.9 to 99.3 per cent of those calculated from the weights of H2C204.Hz0 used in preparing the solutions.
Gasometric Determination of CO2 from Standard Oxalic Acid Solutions.
Of oxalic acid solutions standardized as described above, por- tions of 1.000 or 2.000 cc. were pipetted into the Van Slyke-Neil1 apparatus and brought to 6.0 cc. volume by addition of 2 cc. of 1 N sulfuric acid and water. Permanganate was then added and the analysis was completed as described above for serum deter- minations. The results are given in Table 11. They show that under the conditions employed the yield of COZ is 99.4 per cent of theoretical. Apparently the oxidation at room temperature does not go quite to completion, but the yield of COz is so nearly quantitative and so constant that the slight shortage can be allowed for in calculations without introducing an error. In com- puting all the factors of Table I we have accordingly multiplied by 1.006 the theoretical ratio of oxalic acid or calcium to the COZ formed by permanganate oxidation.
In the experiments of 5-31-27 and 6-2-27 MnS04 was added to the KMnOd to catalyze the reaction if possible. No perceptible difference in the yields was noticed. In the experiments of 7-25-27, and 7-26-27, the samples were first pipetted into 15 cc. centrifuge tubes and washed into the cup of the apparatus as outlined in the description of the method. This check showed the washing to be quantitative. In the experiment of 2-16-29 the solution analyzed was made by dissolving in 2 N sulfuric acid weighed amounts of cal- cium oxalate. The latter had been precipitated by addition of oxalic acid to an excess of thrice washed calcium carbonate dis- solved in HCl and acetic acid. The precipitated calcium oxalate was washed twelve to fifteen times, until the washings were free from calcium and chloride and oxalate, then dried at 60” in a vacuum oven to constant weight. This gave quite an analytically
especially if the precipitate is stirred with each successive portion of washing solution. The usual ammonia water was used for these washings. The latter in the a series were carried out as described above for routine serum analysis, with minimum dis-
turbance of the precipitate, while in the b series the precipitate was stirred up with each portion of washing fluid.
Interference of Serum Proteins with Complete Precipitation of Calcium Oxalate.
Calcium was determined in each of 5 sera by two procedures. (a) The analysis was carried out as described above, with pre-
liminary removal of the proteins by means of trichloroacetic acid. Portions of 10 cc. of filtrate, equivalent to 2 cc. of serum, were used for the analyses.
TABLE III.
E.ffect of Repeated Washing of Calcium Oxalate Precipitate, (a) with and (b) without Stirring. Analyses of Known Calcium Solution.
No. of washings with 3 cc. portions of 2
cent ammonia. per
Calcium per 100 cc.
Present. Found. Error.
(a) 3 (b) 3
m?.
10.02
mg. per cent
10.13 +1.1
9.78 -2.4
(a) 6 9.91 -1.1
(b) 6 8.79 -12.3
(4 9 9.78 -2.4
(b) Q 7.64 -23.8
(a) 12
(b) 12 9.68 6.09
-3.4 -30.7
(6) The calcium oxalate was precipitated in the presence of the serum proteins. In each analysis 2 cc. of serum in a 15 cc. centri- fuge tube were diluted to 5 cc. 1 cc. of ammonium oxalate solu- tion was added, and the mixture was left overnight to precipitate. The washing of the precipitate and subsequent steps of the analy- ses were all carried out according to the routine procedure, except the blank for determination of the c correction. For the blank a sample of serum was treated in every way as in the main analysis, except that in place of 1 cc. of ammonium oxalate 1 cc. of 2 per cent NaCl solution was added. The salt solution redissolved globulin that precipitated when the serum was diluted. The pl -
p2 pressure reading obtained in this blank analysis included not only COz from the reagents, but also a slight amount from oxida- tion of traces of serum organic matter which adhered to the walls of the centrifuge tube even after the two washings with ammonia. Such organic matter was sufficient, to increase the value by 1 to 5 mm., when the gas volume was read at 0.5 cc.
That the low results of precipitation in the presence of the serum proteins (fourth column of Table IV) are due to incomplete Brecipitation of calcium oxalate was shown as follows: A 20 cc. sample of each serum was placed in a large centrifuge tube, 30 cc. of water were added, then 10 cc. of saturated ammonium oxalate
TABLE IV.
Comparison of Serum Calcium Precipitation Performed with and without Preliminary Removal o.f Proteins.
Analysis No. Species.
1 Horse. 2 ox. 3 Man 4 ox. 5 “
Ca pptd. in triohloro-
acetic acid filtrate.
Ca pptd. in diluted 8Wlnn.
A
Ca pptd. in filtrate from A after re-
moval of pm-
YY.
Total Ca ob- tained in di- luted serum
and in filtrate.
A+B
mg. per cent mg. per cent n&g. per cent ng. per cent
solution. The calcium oxalate was allowed to precipitate over- night. The mixture was then centrifuged. The supernatant diluted serum was poured through an ashless filter into a 100 cc. volumetric flask. The transfer was made more nearly quantita- tive by washing the filter. To the filtrate 20 cc. of 20 per cent trichloroacetic acid were added, the mixture was further diluted to 100 cc., let stand a half hour, and then filtered from the protein coagulum. 10 cc. portions of the protein-free filtrate were trans- ferred to centrifuge tubes and brought with sodium acetate and ammonia to pH 5.0, as in the routine method described above.
A precipitate of calcium oxalate formed, proving that the pre- ceding calcium precipitation in the presence of the serum proteins had been incomplete.
The precipitate was washed and determined gasometrically as described for the routine procedure. The amounts of extra calcium recovered are shown in the fifth column of Table IV. Added to the amounts obtained in the first precipitate they yield, except in Analysis 5, total calcium values as near as could be ex- pected to those obtained by the routine single precipitation with preliminary removal of proteins (compare third and last columns).
Micro Analyses oj Standard Calcium Solutions by Gasometric and Titrimetric Methods.
As stated above, the precision of micro calcium methods rests upon details, of which the most important appears to be the technique of washing the precipitate. Before we settled upon the procedure described above for the routine method, we performed some 600 gasometric and titrimetric analyses under varying con- ditions of precipitation and washing. A few illustrative results are given in Table V.
The standard calcium solutions were made from reprecipitated calcium carbonate which was thoroughlywashed and finally dried at 280”. Standard solutions were made from weighed portions, which were dissolved with a slight excess of hydrochloric acid. The accuracy of the standard solutions was controlled by gravi- metric macro analyses, in which the calcium was precipitated as oxalate and weighed as ignited CaO in platinum crucibles.
The micro gasometric and volumetric analyses were carried out in triplicate on solutions containing, as does blood serum, between 8 and 10 mg. of calcium per 100 cc. The samples t.aken varied between 1 and 5 cc. The addition of ammonium oxalate was carried out as in the routine procedure previously outlined. A series of selected but representative results is given in Table V. The time allowed for precipitation ranged from 1 to 24 hours. The washings were made with portions of the 2 per cent ammonia water which were varied in number and volume.
The procedure finally adopted for routine serum calcium deter- minations, in which the washing is carried out with two portions of 3 cc. each of the dilute ammonia, was found to give the best re- sults. With this technique the micro analyses showed maximum deviations of =t3 per cent from the amount of calcium present, plus and minus deviations being about equally frequent. The
great majority of deviations were within fl per cent of the amount of calcium present.
Whatever washing technique was employed, titrimetric and gasometric analyses of precipitates similarly handled usually agreed within ~1 per cent, indicating that with the amounts of
TABLE VI.
Comparison of Gasometric and Titrimetric Analyses of Serum.
material used, the error involved in the fural analysis by either method is unimportant compared with the errors involved in prior handling of the calcium oxalate precipitate.
Comparison of Gasometric and Titrimetric Calcium Determinations in Blood Serum.
The determinations were carried out according to the method described in this paper, except that in the titrimetric analyses the
final solution of calcium oxalate, instead of being cooled and transferred to the Van Slyke-Neil1 apparatus, was titrated hot in its centrifuge tube with 0.01 N or 0.005 N permanganate. The results of a series of parallel analyses are given in Table VI.
SUMMARY,
A method is described, whereby oxalic acid is quantitatively estimated by oxidation with an excess of permanganate in the Van Slyke-Neil1 apparatus, the evolved CO2 being measured manometrically.
Applications of the method to calcium determinations in general, and to serum micro calcium determinations in particular, have been developed.
In connection with the latter it has been shown that removal of the serum proteins is a necessary preliminary to complete pre- cipitation of calcium as oxalate.
BIBLIOGRAPHY.
Clark, E. P., and Collip, J. B., J. Bid. Chem., 63,461 (1925). Clark, G.W., J. Biol. Chem., 49,487 (1921). Halverson, J. O., and Bergeim, O., J. Biol. Chem., 32,158 (1917). Hamilton, B., J. Bid. Chem., 66,101 (1925). Hulett, G. A., and Bonner, W. D., J. Am. Chem. Sot., 31,390 (1909). Kramer, B., andTisdal1, F. F., J. BioZ. Chem., 47,475 (1921). Rothwell, C. S., J. BioZ. Chem., 74,257 (1927). Sendroy, J., Jr., andHastings, A. B., b. Biol. Chem., 82,197 (1929). Shohl, A. T., J. BioZ. Chem., 60,527 (1922). Trevan, J. W., and Bainbridge, I-1. W., Biochem. J., 20,423 (1926). Van Slyke, D. D., J. BioZ. Chem., 71,235 (1927). Van Slyke, D. D., and Neill, J. M., J. BioZ. Chem., 61,523 (1924). Van Slyke, D. D., and Sendroy, J., Jr., Proc. Sot. Exp. BioZ. and Med., 24,
167 (1926). Van Slyke, D. D., and Sendroy, a., Jr., J. BioZ. Chem., 73, 127 (1927).
