A STUDY OF THE ESTIMATION OF SODIUM IN BLOOD SERUM

BY ERIC G. BALL AND JOSEPH F. SADUSK, JR.*

(From the Department of Physiological Chemistry, the Johns Hopkins University, School of Medicine, Baltimore)

(Received for publication, January 21, 1936)

Without doubt the most satisfactory procedure for the quanti- tative determination of small amounts of sodium is that involving its precipitation as uranyl zinc sodium acetate, (UO&ZnNa- (CH,C00)9.6Hz0, first introduced by Barber and Kolthoff (2). Applications of this method to biological material have been numerous, since the triple salt is readily estimated by a variet,y of gravimetric, calorimetric, or volumetric procedures. Of these procedures that which appears to offer the greatest accuracy with the greatest sensitivity is the volumetric one employed by Kolthoff and Lingane (8). This, in brief, involves the reduction of hexava- lent uranium in the triple salt to UxV and some U”‘; the latter is easily oxidized by air at room temperature to the quadrivalent state which is less susceptible to air oxidation. The quadrivalent uranium is then oxidized quantitatively to the hexavalent state with dichromate.

The application of this volumetric procedure to biological mate- rials has not received the attention that the method merits. We therefore present, in the following pages, a method for the deter- mination of sodium in blood serum based on these principles. Accurate results are obtainable on 0.2 ml. samples of serum. In proof we present parallel determinations on 10 to 20 ml. samples by the classical chloroplatinate method. For comparison we also present results obtained by the gravimetric method for estimating the uranium triple salt as described by Butler and Tuthill (3) and the older pyroantimonate method of Kramer and Gittleman (9). A comparison is also made of the values obtained on trichloroacetic acid filtrates of serum and ashed samples.

* Fellow of the Henry Strong Denison Medical Foundation, 1934-35. 661

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662 Estimation of Na in Blood Serum

Reagents- Uranyl zinc acetate reagent, Barber and Kolthoff (2). Solution

A: To 75 gm. of uranyl acetate (2HnO) add 400 ml. of water and 13 ml. of glacial acetic acid. Solution B: To 350 gm. of zinc ace- tate (3HzO) add 400 ml. of water and 10 ml. of glacial acetic acid. Heat to dissolve and mix the two solutions while hot. The result- ing mixture should be saturated at room temperature with uranyl zinc sodium acetate, excess solid being present. An interval of several days before use is advisable. Shake and filter each time immediately before using.

Glacial acetic acid, Salit (13). Saturate glacial acetic acid with the triple salt. Shake and filter each time immediately before using.

Ferric sulfate, 5 per cent. 5 gm. in 100 ml. of 2 N H&Sod. Add dilute KMn04 solution dropwise till a faint pink tinge appears in order to insure the absence of ferrous iron. The excess permanga- nate is now destroyed by boiling the solution vigorously for a few minutes.

Barium diphenylaminesulfonate, 0.2 per cent. An aqueous solution of the salt supplied by the Eastman Kodak Company.

Trichloroacetic acid, 30 per cent. Dissolve 30 gm. of Na-free acid in water and dilute to a total volume of 100 ml.

Potassium dichromate, 0.1 N and 0.025 N. Dissolve 4.901 gm. of pure salt in water and dilute to 1 liter to obtain an exactly 0.1 N solution. Prepare the weaker solution by dilution. Solutions used in the present work were prepared from salt twice recrystal- lized and dried to constant weight at 140”.

Amalgamated zinc. Dissolve 2.5 gm. of mercury in 25 ml. of HNOS(l:l) and add 100 ml. of water and 250 gm. of C.P. granu- lated zinc (20 to 30 mesh, low in iron). Shake vigorously for several minutes. Wash thoroughly by decantation with water and air-dry.

Phosphoric acid, C.P. 85 per cent. Sulfuric acid, 4 N and 2 N. Caprylic alcohol. The various sodium chloride solutions employed were made

from Kahlbaum’s best grade salt (Zur AnaZyyse mit Guruntieschein) dried at 120”.

Jones Reductor-Attempts to reduce the hexavalent uranium

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of the triple salt completely to the quadrivalent or lower state by the action of titanous salts or plain zinc in acid solutions were unsatisfactory. Employment of a Jones reductor gave highly satisfactory results and, since this apparatus is extremely simple in construction, we do not hesitate to introduce it into biological work.

Construction-In brief its construction is as follows: To one end of a glass tube, 32 cm. long and 1 cm. in internal diameter, attach a glass stop-cock with a narrow bore delivery tube of 16 cm. length. To the other end seal an 8 cm. length of tubing, 2.5 cm. in internal diameter; thus forming a cup of about 40 ml. capacity into which solutions to be reduced are easily poured. The apparatus is now ready to be filled. With a few glass beads as a foundation place a layer of glass wool in the closed end of the tube, and follow this with a layer of asbestos about 1 mm. thick. This asbestos layer must be thick enough to keep back the zinc dust, and thin enough to permit free passage of solutions. Amalgamated zinc prepared as described above is now poured into the tube to give a column 25 cm. in height. A thin layer of glass wool now completes the construction.

Operation-The delivery tube of the reductor is inserted into an Erlenmeyer flask closed by a a-hole rubber stopper. Suction is applied to the flask by means of the remaining aperture. The reductor is then cleaned by drawing through it 300 ml. of 2 N sulfuric acid. 100 ml. portions of acid drawn through after this should show no reducing action towards permanganate. If the zinc contains impurities, e.g. iron, a positive test may be encoun- tered. In this case a blank value must be obtained on a volume of solution similar to that employed in the determination and subse- quent results corrected accordingly. The ordinary C.P. grades of zinc with low iron content used in this work gave no blank correc- tions. The solution to be reduced is now poured into the bell of the reductor and suction applied until the column of zinc is barely covered with residual solution. Successive portions of 2 N sulfuric acid are now drawn through to transfer quantitatively the reduced solution to the flask, each time leaving the zinc column barely covered with liquid. After changing the receiving flask the apparatus is immediately ready for the reduction of another solu- tion. When it is not in use, e.g. overnight, the sulfuric acid is

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664 Estimation of Na in Blood Serum

removed from the reductor by drawing water through it and storing with sufficient water to keep the zinc column well covered. Before use the water is replaced by drawing through the reductor 50 ml. of 2 N sulfuric acid. The zinc column should be kept immersed at all times, the level of fluid never being allowed to fall below the top of the zinc column whether the apparatus is in use or stored. If the zinc is allowed to become dry, 200 to 300 ml. of 2 N HzS04 should be run through the apparatus before using it again. For further details concerning the Jones reductor the reader should consult standard texts, e.g. (6).

All volumetric apparatus employed were calibrated. Pipettes used for measuring serum were intercalibrated by weighing the volume of serum delivered and the results for the different methods were corrected accordingly.

Procedure

Removal of Protein. Ash&g LTo 1 ml. of serum contained in a platinum or a silica crucible add 1 ml. of 4 N sulfuric acid. Evap- orate on a steam bath for about $ hour, when a black residue is left. Place in a cold muffle furnace and gradually raise the temperature to 600”; avoid higher temperatures. Heat until a white ash re- sults. Employing an electric furnace, calibrated with a thermo- couple, we have found it convenient to start the furnace at the end of the day and allow it to run overnight. With the rheostat set for 600” the cold furnace has warmed up gradually enough to prevent spattering, and ashing is complete by morning. For 0.2 ml. samples of serum the procedure is the same except 0.5 ml. of 4 N H&S04 is used.

Precipitation-To 1 ml. of serum contained in a 15 ml. centrifuge tube add exactly 5 ml. of distilled water and a fraction of a drop of caprylic alcohol. (The alcohol is added to prevent subsequent adherence of precipitated protein to the sides of the tube and as shown by control experiments has no effect per se on the results.) Mix thoroughly and add 1 ml. of 30 per cent trichloroacetic acid, drop by drop, with constant stirring. Centrifuge down the precipitated protein and draw off the supernatant fluid, of which a

1 Attempts to employ wet ashing with sulfuric and nitric acids resulted in low and erratic results when the sodium was subsequently determined by the volumetric uranium procedure.

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5 ml. aliquot is taken for analysis. The procedure for 0.2 ml. of serum is the same, the total volume being kept at 7 ml. by the addition of more water, 1 ml. of trichloroacetic acid still being employed. In working with 0.2 ml. samples we have preferred to obtain these by adding 0.5 ml. of serum to 2 ml. of water and taking 1 ml. of the mixture.

Determination of Sodium. Volumetric, on 0.2 Ml. of Serum-If the serum is ashed, add to the residue in the crucible 1 drop of 2 N sulfuric acid and transfer with two 0.5 ml. portions of water to a 15 ml. Pyrex centrifuge tube. Tubes should be kept clean by soaking in cleaning solution and well rinsed before use. Rinse the crucible with small portions of the uranium reagent. Crucibles that pour poorly may be smeared below the lip with Vaseline. If a filtrate is employed, evaporate 5 ml. portions to incipient dryness in a crucible in order to adjust volume relations and proceed as outlined above.2

To the solution in the centrifuge tube add uranyl zinc acetate reagent to give a total of 10 ml. pouring down into the tube so as to wet the entire inner surface. Stir vigorously with a motor stirrer (see under “Results”) for 10 minutes. Centrifuge down the sodium salt and decant the supernatant fluid, rotating the tube so that the entire inner surface is wetted. Allow the tube to stand inverted over filter paper for 5 minutes. Wipe the mouth of the tube dry and pour down the sides of the tube 10 ml. of the glacial acetic acid reagent, stirring up the precipitate. Centrifuge, decant, and drain again as above. Dissolve the precipitate in 15 ml. of 2 N sulfuric acid and transfer to the Jones reductor. Draw the solution through the reductor as described earlier at a rate such that the operation will be complete in about 45 seconds. Flush the reduc- tor by drawing through it successively four 20 ml. portions of 2 N sulfuric acid, at the rate of approximately 40 ml. per minute. Dis- connect the receiving flask from the reductor and draw air through the solution for 5 minutes. Add in the order named 5 ml. of a 5 per cent ferric sulfate solution, 5 ml. of 85 per cent phosphoric acid,

* Evaporation of the filtrate in the centrifuge tube in which the sodium precipitation is carried out leads to low results. We attribute this to a “dirtying” of the walls of the tube during evaporation, which in some way interferes with the complete precipitation of the sodium. Clean tubes are essential to good results. This is an example of the attention that must be paid throughout to details, when employing small amounts of sodium.

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666 Estimation of Na in Blood Serum

and 5 drops of barium diphenylaminesulfonate indicator,3 swirling the flask vigorously after each addition. Titrate with 0.025 N potassium dichromate until a violet color which persists for at least 30 seconds is obtained. Subtract 0.06 ml. from the final value for the indicator correction (14).

Culculations-Ashe.d sample: Mg. of Na/lOO ml. = (ml. of 0.025 N K2Cr20, - 0.06 ml.) X- 0.0958 X (100/0.2). For the filtrate, calculate as above and multiply by 8.

Volumetric, on 1.0 Ml. of Serum-If an ashed sample is em- ployed, add 1 drop of 2 N sulfuric acid to the contents of the crucible and transfer to a well cleaned 50 ml. conical bottom centrifuge tube, using a total of 5 ml. of water. For filtrates a 5 ml. aliquot is used directly. Add 45 ml. of the uranyl zinc acetate reagent and stir by hand for 2 minutes after the appearance of a precipitate. Allow to stand for 15 minutes. Separate the precipi- tate from the excess reagent by centrifuging, decanting, and wash- ing with 25 ml. of the glacial acetic acid reagent, as described above for the smaller serum sample. Dissolve the precipitate in 30 ml. of 2 N sulfuric acid and draw through the Jones reductor. Flush with five 20 ml. portions of 2 N sulfuric acid. Observe the pre- cautions and rates of flow previously described. Aerate the reduced solution for 5 minutes. Add in order and with shaking 15 ml. of ferric sulfate reagent, 5 ml. of phosphoric acid, and 5 drops of indicator solution. Titrate with 0.1 N dichromate. No indi- cator correction is necessary with this larger amount of sodium.

Calculations. Ashed Sample-Mg. of Na/lOO ml. = ml. of 0.1 N K2Cr20, X 0.3833 X 100. For the filtrate, calculate as above and multiply by %.

It is unnecessary to remove phosphate before determining sodium by this method. Assuming the phosphate to be precipi- tated as UOz.HPOd, 100 ml. of serum containing a total of 10 mg. of P will bind only as much uranium as 2.5 mg. of Na. This is an error of about 0.7 per cent for serum containing 350 mg. of Na per 100 ml. Analysis of solutions of known Na content and containing as much as 14 mg. of P per 100 ml. gave theoretical results (see Table IV). To check this point further we have tried

a The ferric salt and phosphoric acid are added in order to catalyze the slow reaction between dichromate and the indicator; a sharper end-point results. (See reference (8) for further details.)

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adding iron as recommended by Hald (5) for the removal of phosphate. Low results were obtained. The insoluble iron precipitate, when dissolved in acid, yielded a positive test for sodium. The fault may be ours in using an excess of iron, which the author states introduces difficulties in the solution of the ash. This point was not further investigated by us.

For comparison we have employed three other methods. These require brief comment. We have taken the classical Lindo-Glad- ding chloroplatinate method as our bed-rock of accuracy. The procedure used was that outlined by the Association of Official Agricultural Chemists (11). All samples were ashed in a muffle furnace as described above, 0.5 ml. of 4 N HzS04 being used to each ml. of serum. For the removal of calcium, magnesium, and phosphate we first followed the short cut suggested by Mackay and Butler in Peters and Van Slyke (12). This procedure in our hands failed to remove completely these interfering salts and we returned to the longer but more’satisfactory stepwise precipitation of the official method employing also ammonium oxalate. In order to obtain sodium-free ammonium hydroxide for use in this method we chose to prepare this reagent by passing tank ammonia gas into distilled water contained in a paraflin-coated Pyrex flask. The pyroantimonate method employed was t,hat of Kramer and Gittleman as modified by Kerr (7). Ashed samples of serum were employed. Lastly we have employed the gravimetric method of estimating the triple uranium salt as outlined by Butler and Tuthill (3). All samples were ashed in a muffle furnace. Diffi- culty encountered by other workers (5, 13) in maintaining the alcohol wash solution saturated with the triple salt was also our experience.

Results

We have first investigated the effect of the length of time allowed for the precipitate to settle after formation and the type of stirring during precipitation on the accuracy of the results. The majority of previous workers, following Barber and Kolthoff, have allowed 1 hour to elapse for complete precipitation. Results given in Table I show that 15 minutes are sufficient time for complete precipitation of 1.0 to 5.0 mg. of sodium. For 0.5 mg. of sodium low results were obtained regardless of the elapsed time. These results were

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668 Estimation of Na in Blood Serum

obtained by the volumetric procedure outlined above for serum, hand stirring for 2 minutes being employed throughout. If, however, the precipitation of this small amount of sodium is carried out, by motor stirring instead of hand stirring, a marked

Anal :l/s is of Sodium Chloride Solutions by Volumetric Procedure

I 15 min.* T- 60 min.’

Na taken Na found NE found Difference

ma. 0.500

1.00

2.00

5.00

0.1 i-i KeCreOr Difference 0.1 N

K&ho7 _ - ml. per cent ml.

1.275 -2.2 1.265 1.270 -2.6 1.275 2.60 0.0 2.59 2.63 +1.0 2.57 5.27 -1.0 5.23 5.23 +0:5 5.22

13.03 -0.2 13.02 13.01 -0.2 13.02

* Time that sodium precipitate was allowed to settle after formation.

ml.

0.489

0.437 1.00 1.01 2.02 2.01 4.99 4.99

--

0245

per cent

-3.0 0.489 -2.2 0.99 -1.0 0.99 -1.0 2.01 +0.5 2.00 0.0 4.99 -0.2 4.99 -0.2

TABLE I

TABLE II Effect of Stirring on Accuracy of Volumetric Procedure When Determining

Small Quantities of Sodium In each experiment 0.500 mg. of Na was the amount taken.

0.025 N K&r&h

Motor stirring

ml.

5.25 5.23 5.31 5.30 5.25 5.30

Na found DiI%PXV33

0227

0.495

0.503 0.502 0.497 0.502

pa Cent -0.6 -1.0 +0.6 +0.4 -0.6 +0.4

.-

.-

-

0.026 N KZC~207

ml. mg. per cent

5.16 0.489 -2.2 5.21 0.493 -1.4 5.15 0.488 -2.4 5.18 0.490 -2.0

Hand stirring

Na found Diierence

improvement in accuracy is obtained. Results of such an experi- ment are given in Table II. It is, therefore, apparent that stirring is more important than prolonged standing and we recommend motor stirring for 10 minutes and no further standing when dealing

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with 0.5 mg. or smaller quantities of sodium. Temperature con- trol appears to be of minor importance. We have worked at normal room temperature, taking care only to prevent gross fluctuations during precipitation and washing.

TABLE III Sodium Content of One Sample of Dog Serum Analyzed by Various Methods

The results are expressed in mg. of Na per 100 ml. of serum. -

-_ serum ash serum mrate

pyre- anti-

wmatc uranium, volumetrio -I kavi-

netrio

1 ml.

, ( I

lml. -

.5 ml. ’ I _-

20 ml. 2 ml. D.2 ml - lml.

-i-- 4 ml.

347 348 349 350

351 348

349 349

347 354 358 354 344 350 358 356

- 2ml -

355 352

1 ml.t

351 351

349 350

347 349

345 347

365 363

353 355 354 356 352 356 358 354

347 356 352 354

353 358 355 360

357 356

356 355

355 354 ~ 356 -

353 351

- 353 -

367 369

- _

Average. 348 349 349 349 349 353 355 -

* Procedure run through as for 1 ml. samples of serum. t Serum added to 5 per cent CCLCOOH. See discussion of results.

366

In Table III are summarized the results obtained on one sample of dog serum analyzed by various methods. Ashed samples gave practically identical values when analyzed by the macrochloro-

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670 Estimation of Na in Blood Serum

platinate, pyroantimonate, gravimetric, or volumetric uranium procedures. The reproducibility of the uranium procedure is attested to by the number of determinations all agreeing to within

TABLE IV Sodium Content of Various Samples of Sera

The results are expressed in mg. of Na per 100 ml. of serum.

Beef. .............

Cat. ..............

Normal Human 1.

“ “ 2.

“ “ 3.

“ “ 4.

Human 5t.. . . .

Artificial serum&.

. . . . . .

. .

. . . . .

-

-

I Pi -

--

-

LO ml.

339 340 367 368

350 350

2nd.

339

339

356 354 357 346

316* 324*

308*

350 350

-I- t

1

.-

.-

335 339 360 365

324 327 316 322 320 327 307 306 284 2% 348 349

Volu- metric

0.2 ml.

336 335

345 347 371 372

D.2 ml.

341 346

325 331 327 328

331 328 336 335 313 313 298 296 350 350

7--

Serum filtrate

1 ml.

- ,

-

* 1 ml. samples. t Patient had received glucose intravenously. $ A non-protein solution containing, per 100 ml., 350 mg. of Na, 21 mg.

of K, 11 mg. of Ca, 3.5 mg. of Mg, 14 mg. of P. All manipulations used for removal of protein from serum were also applied to this solution.

fl per cent. The results on filtrates are not as satisfactory. Depending on the mode of precipitation and the ratio of serum volume to total filtrate volume, results varied from 1 to 5 per cent higher than those found for ashed samples. For example the

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E. G. Ball and J. F. Sadusk, Jr.

analyses reported in the last column of Table III were obtained on a filtrate prepared by adding 1 ml. of serum to 6 ml. of 5 per cent trichloroacetic acid. The results are 5 per cent too high. The analyses reported under the other columns headed “Filtrate” were obtained on a filtrate prepared by diluting the serum sample (0.2 to 1.0 ml.) to 6 ml. with water and adding 1 ml. of 30 per cent trichloroacetic acid. The results obtained here are only 1 to 2 per cent too high, the best agreement with the ashed values being obtained on the 0.2 ml. samples, where the serum dilution at the time of precipitation is greatest. Filtrates prepared with various other strengths of trichloroacetic acid and modifications gave values intermediate to those described above. Best results are therefore obtained on a filtrate prepared with 30 per cent trichloro- acetic acid, according to the technique described under “Proce- dure.” This peculiar effect will be discussed later.

Table IV contains the results obtained on various samples of sera. The 6ndings are similar to those reported for dog serum in Table III. The values given by filtrates again run from 1 to 3 per cent too high. The results obtained by the pyroantimonate method are in good agreement with those obtained by the uranium and macrochloroplatinate methods and show that this older method is reliable if applied to ashed samples. One of us (1) has previously shown in the analysis of pancreatic juice that the pyroantimonate method gives values in agreement with those obtained by the macrochloroplatinate procedure.

DISCUSSION

The results obtained indicate that sodium may be determined in blood serum by the uranium procedure with an error of less than 1 per cent, if ashed samples are employed. Ashing in a muffle furnace is the procedure of choice and in our hands has required less attention from the analyst than any other method including filtrates. There is little to choose between the volumetric and gravimetric uranium procedures when 1 ml. samples are available. In fact, owing to its greater simplicitywe are inclined to recommend the gravimetric method for samples of this size, though we feel that the volumetric procedure will yield more reproducible values. Occasionally the gravimetric method has yielded duplicates which do not agree as well as we would like; this has never been the case

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672 Estimation of Na in Blood Serum

with the volumetric procedure. For samples smaller than 1 ml. we recommend the volumetric uranium procedure.

Filtrates may be employed, if the highest accuracy is not re- quired. Results will be from 1 to 3 per cent too high, if the tech- nique outlined under “Procedure” is followed. This positive error has been so consistently encountered we feel that an empirical correction may be applied to the results, though we do not whole heartedly recommend such practices. The average positive error with use of filtrates from 1 ml. and 0.2 ml. samples has been respectively 2.8 f 1.0 per cent and 1.2 f 0.5 per cent. If a cor- rection based on these figures is applied, the preparation of the iiltrate must be as directed here, the trichloroacetic acid must be proved Na-free as described below, and the volumetric procedure must be employed.

We have endeavored to eliminate this annoying discrepancy between filtrates and ashed samples by a stepwise investigation of all possible sources of error. Tests for sodium in the trichloro- acetic acid were negative. A solution approximating serum in its inorganic composition and containing known amounts of sodium (see Table IV) was run through the trichloroacetic acid procedure used to precipitate the proteins in serum. Subsequent analysis for sodium gave values identical with untreated samples and with the theoretical. Trichloroacetic acid was found to cause no interference in the volumetric estimation of the uranium; known uranium solutions with and without trichloroacetic acid gave identical values. It was thought possible that the filtrates might contain serum organic substances not precipitated by trichloro- acetic acid but precipitated as uranium salts along with the sodium. Filtrates were therefore ashed before analyzing for sodium. High results were still obtained. Investigation of other minor details proved as futile. The only explanation we csn offer for this phenomenon is that of volume displacement; i.e., the precipitated protein occupies a definite volume which actually diminishes the volume of the filtrate and so increases the concen- tration of the solutes, In support of this view are the facts first, that the error diminishes as the serum volume diminishes in rela- tion to the total filtrate volume, and second, that the mode of precipitation which gives the smallest bulk of precipitated protein yields results nearest to the true value.

Upon looking over the literature it was found that other workers

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E. G. Ball and J. F. Sadusk, Jr. 673

had encountered a similar experience when employing trichloro- acetic acid filtrates. Van Slyke, Hiller, and Berthelsen (15) found serum filtrates yielded values 4 per cent higher than ashed serum samples, when analyzed for total base, and explained the’ error as due to volume displacement. Grigaut and Boutroux (4) report a +3 per cent error in their sodium determinations on serum filtrates and Oberst (10) found even a greater error, when determining sodium on filtrates of red cells where the protein bulk is large. A similar explanation of their results is offered by these workers. We are not aware of any cases in which a volume displacement effect has been noted on other than trichloroacetic acid filtrates or in which sodium was not involved in the determination. A careful investigation of this aspect with regard to other serum constituents would be of interest though probably difficult to achieve, especially in the case of organic material. Chloride offers the best opportu- nity. Wilson and Ball (16) found tungstate filtrates of serum and whole blood to give chloride values in good agreement with ashed samples determined gravimetrically. However, compensating errors (e.g., the possible binding of the negative chloride ion as well as tungstate by the precipitated protein) which were not investi- gated might account for the agreement in this case.

SUMMARY

There is described a volumetric procedure for the determination of sodium in as little as 0.2 ml. of serum. The method is based on the precipitation of sodium as uranyl zinc sodium acetate, the reduction of the uranium in the precipitate to the quadrivalent state, and its subsequent quantitative oxidation with dichromate to the hexavalent condition. Serum ash or titrate may be em- ployed, though results with the latter give values 1 to 3 per cent too high, an effect ascribed to a volume displacement by the precipitated protein. Results on ashed samples agreed within 1 per cent with those found by the macrochloroplatinate procedure. The gravimetric uranium and pyroantimonate methods were also employed for the sake of comparison and found to be entirely reliable.

BIBLIOGRAPHY

1. Ball, E. G., J. Biol. Chem., 88,433 (1930). 2. Barber, H. H., and Kolthoff, I. M., J. Am. Chem. SOL, 50,1625 (1923). 3. Butler, A. M., and Tuthill, E., J. Biol. Chem., 98,171 (1931).

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Estimation of Na in Blood Serum

4. Grigaut, A., and Boutroux, A., Compt. rend. Sot. biol., 104,872 (1930). 5. Hald, P. M., J. Biol. C&m., 103,471 (1933). 6. Hildebrend, W. F., and Lundell, G. E. F., Appliedinorganicanalysis,

New York, 100 (1929). 7. Kerr, S. E., J. Biol. Chem., 67,689 (1926). 8. Kolthoff, I. M., and Lingane, J. J., J. Am. Chem. Sot., 66,187l (1933). 9. Kramer, B., and Gittleman, I., J. Biol. Chem., 62,353 (1924-25).

10. Gberst, F. W., J. Biol. Chem., 106, 153 (1935). 11. Official and tentative methods of analysis of the Association of Official

Agricultural Chemists, Washington, 3rd edition, 106 (1930). 12. Peters, J. P., and Van Slyke, D. D., Quantitative clinical chemistry,

Methods, Baltimore (1932). 13. Salit, P. W., J. Biol. Chem., 96,659 (1932). 14. Sarver, L. A., and Kolthoff, I. M., J. Am. Chem. Sot., 63,2906 (1931). 15. Van Slyke, D. D., Hiller, A., and Berthelsen, K. C., J. Biol. Chem., 74,

659 (1927). 16. Wilson, D. W., and Ball, E. G., J. Biol. Chem., 79,221 (1928).

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Eric G. Ball and Joseph F. Sadusk, Jr.SODIUM IN BLOOD SERUM

A STUDY OF THE ESTIMATION OF

1936, 113:661-674.J. Biol. Chem. 

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