a new triple acetate method for sodium de

A NEW TRIPLE ACETATE METHOD FOR SODIUM DE- TERMINATIONS IN BIOLOGICAL MATERIALS

BY PETER WALDEMAR SALIT

(From the Department of Ophthalmology, the State University of Iowa, Iowa City)

(Received for publication, March 28, 1932)

It has been known for years that sodium combines with zinc uranyl acetate or magnesium many1 acetate in a definite ratio to form yellow crystals, soluble in water but only slightly soluble in alcohol. In 1886, Streng (1) studied this compound and employed it for the detection of sodium. He ascribed to it the formula (UOz)aMgNa(CH&00)9.9Hz0. According to Miholic (2) the compound contains only 6 molecules of water. While Blanche- tiere (3) corroborates the statement of Streng, the findings of Barber and Kolthoff (4) agree with those of Miholic. Barber and Kolthoff state that the ratio of the weight of sodium to the weight of the triple acetate (U02)3ZnNa(CH&00)9.6Hz0 is 0.01495:1. Furthermore, Barber and Kolthoff (4, 5), who were the first to employ this compound as a basis for quantitative estimation of sodium, made a thorough study of the influences of various ions on sodium estimations by this method. They state that barium, calcium, magnesium, and ammonium do not interfere; cesium and rubidium likewise do not interfere if present in quantities below 0.1 gm. Lithium, strontium, and organic acids, as oxalic and tartaric acids, do interfere. Potassium interferes if present in large amounts; i.e., 50 mg. per 1 cc. Phosphates and arsenates interfere, but may be removed easily with magnesia mixture.

The reagents employed by these investigators in the quantitative estimation of sodium were (a) uranyl zinc acetate saturated with uranyl zinc sodium acetate, and (b) 95 per cent alcohol saturated with this triple acetate. Sodium was precipitated by the addition of 20 cc. of uranyl zinc acetate to every 2 cc. of the sample. The precipitate was transferred to a glass filter crucible

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Triple Acetate Method for Na

on a suction flask where it was first washed with the 95 per cent alcohol saturated with triple acetate and then with ether. The precipitate was dried to constant weight, and the sodium was estimated by multiplying the total weight by 0.01495.

The results published by Barber and Kolthoff show that the method was satisfactory when applied to samples containing rela- tively large amounts of sodium; i.e., 75 to 494 mg.

Butler and Tuthill (6) applied a slightly modified Barber-Kol- thoff method to urine, and obtained satisfactory results with samples containing as little as 1.5 mg. of sodium. For the removal of phosphates powdered calcium hydroxide was employed. The method was satisfactory for blood serum, but at least 1 cc. was necessary and it had to be digested with concentrated sulfuric acid.

In my experience, the Barber-Kolthoff method gives satisfactory results with quantities employed by Butler and Tuthill, but cannot be satisfactorily used with smaller quantities. Caley (7) tried the method on samples containing 0.1 to 0.5 mg. of sodium with unsatisfactory results.

McCance and Shipp (8) devised a calorimetric method in which the color was developed with potassium ferrocyanide. They obtained satisfactory results with even smaller quantities of sodium than those used by Caley. In the hands of the writer, however, their method failed to give consistent results. Several other workers have experienced difficulties with the method.

From a study of various modifications of the original Barber- Kolthoff method it is apparent that the principal difficulty with this method lies in the solubility of triple acetate in water. Even if the zinc uranyl acetate reagent is saturated with the triple acetate, an addition of 2 cc. of a sample containing about 0.1 mg. of sodium to 10 cc. of the reagent reduces the concentration of the triple acetate to such an extent that none of the sodium comes down as a precipitate. Precipitation in the cold and the use of large amounts of the reagent are the only known means for over- coming this difficulty. However, if the temperature and time are not strictly controlled, refrigeration is of no value. If the temperature is too low, not only the sodium of the sample is precipitated but some of the reagent and practically all the triple acetate with which it is saturated. Furthermore, if the experi-


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P. W. Salit 661

ment is performed in warm weather, some of the precipitate goes into solution during centrifuging and draining.

In our laboratory, where it is desirable to make accurate determinations of sodium in a drop of aqueous or vitreous humor, without being able to run duplicates, a more reliable method is necessary. This has been accomplished by a method in which sodium is precipitated quantitatively from the uranyl zinc acetate reagent, without saturation with triple acetate, at room temperature by the use of alcohol. Furthermore, by a series of experiments, it was found that glacial acetic acid saturated with triple acetate is a far better wash reagent than alcohol saturated with triple acetate. The entire determination is made at room temperature and in detail is as follows:

Major Reagents and Appliances

1. Uranyl zinc acetate reagent as prepared by Butler and Tut- hill (6) but without saturation with triple acetate. Solution A consists of 80 gm. of sodium-free uranyl acetate (c.P., Baker’s Analyzed) and 46 cc. of 30 per cent acetic acid (by volume) plus water to make 520 gm. Solution B consists of 220 gm. of zinc acetate and 23 cc. of 30 per cent acetic acid plus water to make 520 gm. The solutions are made by placing the ingredients in covered beakers and heating over a steam bath with occasional stirring until dissolved. The two solutions are mixed while hot, allowed to stand for 24 hours, then filtered into a bottle. Shortly before using, the necessary quantity is removed with a pipette and again filtered. The excess is replaced in the bottle.

2. Absolute alcohol or 95 per cent alcohol. 3. Glacial acetic acid (c.P., Baker’s Analyzed, special, 99.6

to 99.7 per cent) saturated with sodium uranyl zinc acetate. Approximately 1 gm. of triple acetate is placed in a bottle, mois- tened with the least possible quantity of distilled water, 1 liter of glacial acetic acid is added, and the contents are well mixed. Before use, the bottle is well shaken and the precipitate allowed to settle. The necessary quantity is then filtered into a large test-tube.

4. A 20 per cent aqueous solution of potassium ferrocyanide (c.P., Baker’s Analyzed). It is filtered if necessary.

5. Standard sodium chloride solution. Exactly 5 gm. of sodium


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chloride (c.P., Baker’s Analyzed) are dissolved in a 500 cc. volumetric flask and made up to volume. 1 cc. of this solution contains 10 mg. of sodium chloride or 3.9345 mg. of sodium.

6. Whatman No. 42 filter paper is used in all filtrations. 7. Stirring rods: No. 1, a fine glass rod about 0.5 mm. in diame-

ter; No. 2, a larger glass rod about 1 mm. in diameter. Each of these is made by heating glass tubing and pulling out into a fine rod; the ends are sealed and flattened.

Minor Reagents (for Sodium Determinations in Biological Materials)

1. Powdered calcium hydroxide (Baker’s Purified). This contains traces of sodium; however, the same quantity is added to both sample and standard and does not affect the end result.

2. A 20 per cent trichloroacetic acid solution (u.s.P., Baker’s or Merck), It is filtered after 24 hours.

3. Concentrated ammonium hydroxide (sp. gr. 0.9, c.P.,

Baker’s Analyzed). 4. A 1 per cent solution of phenolphthalein in absolute alcohol. 5. Perchloric acid (sp. gr. 1.54, Baker’s Analyzed).

General Procedure

Exactly 2 cc. of the sample solution (containing 0.05 to 0.5 mg. of sodium) are delivered into a 15 cc. centrifuge tube. The same quantity of a standard sodium chloride solution, containing approximately the same amount of sodium, is measured into another centrifuge tube of the same size. To each are added 6 cc. of freshly filtered uranyl zinc acetate reagent. With a 1 cc. Mohr pipette graduated into 0.1 cc. exactly 0.3 cc. of absolute alcohol is added to each tube. The alcohol is stirred into the solution with the No. 1 stirring rod. The bulk of the precipitate is allowed to settle into the lower half of the solution, then another 0.3 cc. of alcohol is added to each tube and the stirring repeated without disturbing the precipitate on the bottom. The same stirring rod is used for each tube, without rinsing. After a few minutes another 0.3 cc. of alcohol is added and stirred again. This procedure is repeated four more times, seven times in all, until 2.1 cc. of alcohol have been added. The time intervals are shortened after the second or third addition; however, the process of precipitation consumes at least fr hour. (If the entire quantity of


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P. W. Salit

alcohol is added at once, the results are not satisfactory.) When precipitation is complete, the contents of the tubes are centrifuged for 10 minutes. The supernatant fluid is decanted and the tubes, still held in an inverted position, are placed on coarse filter paper and allowed to drain. The precipitate is washed with 5 cc. of freshly filtered saturated solution of triple acetate in glacial acetic acid. The reagent is delivered from a pipette by gentle blowing; the pipette is pressed against the wall of the tube immediately below its edge while the tube is rotated between the fingers. The precipitate is thoroughly stirred with a No. 2 stirring rod and distributed uniformly throughout the wash reagent. Before trans- ferring the stirring rod to the next tube, it is rinsed with a few drops of wash reagent. After centrifuging, the tubes are drained as before, the mouth of each tube is then wiped with a moist cloth to remove all traces of uranyl zinc acetate reagent, and to each tube is added a drop of glacial acetic acid. The walls of the tubes are rinsed with distilled water from a wash bottle until the solution reaches a point 1 inch below the top. The precipitate is dissolved and evenly distributed throughout the tube by thorough stirring. The contents of the tubes are transferred by means of a small funnel to 50 cc. volumetric flasks. The tubes are filled with water as before; the contents are stirred and transferred to the volumetric flasks. This procedure is repeated once again, and finally the funnel is rinsed, and the volume made up to mark. The contents of each flask are well mixed by shaking. If the amount of sodium lies between 0.1 and 0.2 mg. and the standard contains approximately the same amount, exactly 20 cc. are pipetted from each flask into large test-tubes.l To each tube is added 0.5 cc. of 20 per cent potassium ferrocyanide solution and the contents are well mixed. (The color should never be developed before making up the solution to the desired volume.) After standing 5 minutes, the samples are ready for reading in the calorimeter. If one works with amounts of sodium between 0.2 and 0.3 mg., only 10 cc. of the contents of the volumetric flasks are pipetted into the test-tubes and the volumes are made up to 20 cc. by adding to each tube 10 cc. of distilled water followed by thorough mixing with a stirring rod. When one is working with uncertain quanti-

1 Test-tubes, 6 X 1 inch, are preferable.


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664 Triple Acetate Method for Na

ties of sodium, the concentration of sodium (or uranium) in each sample must be ascertained by trial experiments. 2 or 3 cc. of each solution are placed in small test-tubes and a drop of potassium ferrocyanide solution is added. From this a rough estimate may be made as to the extent each should be diluted before developing the color. Thus the contents of one tube may be made up of 5 cc. of distilled water and 15 cc. of sample solution, another may be made up of equal amounts of distilled water and sample solution, while a third one may need no dilution. If the final volumes are the same, the dilutions of samples and standard are expressed in the calculation as follows:

15, x 0.39345 x 10

16.4 X 0.1 X 15b = mg. Na per 1 cc. urine

where 15, is the reading of the standard; 16.4, the reading of the sample; 0.39345, mg. of sodium in the standard; 0.1, cc. of urine in sample; 10, cc. of standard solution contained in the 20 cc.; 15b, cc. of the sample solution contained in the 20 cc.

If many sodium determinations are made simultaneously, color should never be developed in more than six samples at the same time. For a new series of samples another portion of the standard solution contained in the 50 cc. volumetric flask is used and in this the color is developed. Since the intensity of the color under- goes a gradual change, it is well to make fewer readings than to make many readings at the expense of time. A rise in temperature accelerates this color change, consequently the light in the illuminating chamber should be turned off during preparation of the next sample.

Application of Method to Biological Materials

Blood Serum-To 0.5 cc. of serum diluted to 5 cc. in a 15 cc. centrifuge tube are added 5 cc. of 20 per cent trichloroacetic acid solution. At first the acid must be added drop by drop under vigorous stirring, then more rapidly. After standing a few minutes the tube is centrifuged. Exactly 2 cc. of the supernatant fluid are placed in another 15 cc. centrifuge tube. At the same time the standard is prepared as follows: 5 cc. of standard sodium chloride stock solution (1 cc. of which equals 3.9345 mg. of sodium) are diluted to 100 cc. in a volumetric flask with 10 per cent trichloro-


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acetic acid solution, and 2 cc. (0.39345 mg. of Na) are placed in a 15 cc. centrifuge tube. To the sample and the standard are added 6 cc. of freshly filtered uranyl zinc acetate reagent and the treatment is continued as outlined under “General procedure.” Since the sample contains more than 0.3 mg. of sodium, only 10 cc. of sample and 10 cc. of standard solution are pipetted into the test-tubes; they are made up to 20 cc. by adding 10 cc. of distilled wat,er before developing the color.

Calculation

R standard X 0.39345 X 100 R sample X 0.1

= mg. Na per 100.~~. serum

Each experiment usually consisted of two determinations carried out as follows: The blood serum was divided into two parts. After adding to one a known quantity of standard sodium chloride solution and precipitating the protein in both by trichloroacetic acid, 2 cc. of each were employed. The results are given in Table I.

Whole Blood (from Finger Tip)-In this procedure only 0.2 cc. of blood is necessary. This amount of blood is collected in a serological pipette and discharged into a 15 cc. centrifuge tube, containing exactly 3 cc. of distilled water. The protein is precipitated by the slow addition, with constant stirring, of 0.8 cc. of 20 per cent trichloroacetic acid. The tube is centrifuged, and 2 cc. of the supernatant fluid are used to obtain the precipitate as described under “General procedure.” The data obtained are given in Table II.

Urine-Different urines vary widely in sodium content, and for this reason duplicate or triplicate analyses with varying amounts of urine are run. The bulk of phosphates, uric acid, etc., are first precipitated by allowing the urine to stand in a refrigerator for several hours. After filtering, 3 cc. of the filtrate are placed in a 15 cc. centrifuge tube containing about 0.3 gm. of dry calcium hydroxide. At the same time 3 cc. of a standard sodium chloride solution, prepared by diluting 15 cc. of the standard sodium chloride stock solution to 100 cc. and containing 0.5902 mg. of Na per cc., are pipetted into a similar tube containing 0.3 gm. of calcium hydroxide. The volumes of the standard and sample are made up to 10 cc. by adding to each tube 7 cc. of 1


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Subject NO. Exp~o?-t

1 2 3 4* 5* 6* 1 1 2

TABLE I

Blood Serum

Na in serum Na calculated Ns found

ng. per cent ml. mg.

359.9 0.2780 0.2784 359.9 0.2783 0.2797 357.7 0.2772 0.2720 359.9 0.2783 0.2771 353.2 0.2750 0.2745 353.2 0.2750 0.2720 355.5 0.3745 0.3745 347.1 0.4629 0.4582 347.1 0.4629 0.4611

Recovery

per cent

100.0 100.4 98.1 99.6 99.8 98.9

100.1 99.0 99.6

* These experiments differed from others in the following details: After the protein was precipit@ed in both the sample and the sample plus the standard with trichloroacetic acid in a 0.5:5 ratio, 2 cc. of the supernatant fluid, in a 15 cc. centrifuge tube, were neutralized with 2 cc. of 3 per cent ammonium hydroxide. A small quantity of powdered calcium hydroxide was then added to each sample, including the standard. After allowing the tubes to stand overnight, they were centrifuged, and 2 cc. of the supernatant fluid were used in the precipitation of the sodium uranyl zinc acetate. The results are slightly lower, and the difference may be attributed to the removal of phosphates. It is evident that there is no necessity for the removal of phosphates.

TABLE II

Whole Blood

Subject No. Experiment No.

1 1 2 1 2 2 2 3 2 4 3 1 3 2

Na per 100 cc. blood

WT.

194.1 198.1 199.4 196.7 200.7 209.3 209.3

per cent ammonium hydroxide, and the contents are thoroughly mixed with a small stirring rod. The tubes are allowed to stand at room temperature for at least an hour, during which time the contents are repeatedly stirred. If the tubes stand overnight’,


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P. W. Salit 667

they should be closed with rubber stoppers. After centrifuging, the supernatant fluid of the sample is prepared in triplicate as follows: (1) 1 cc. of the fluid is diluted to 4 cc. with distilled water. This is well mixed, and 2 cc. (0.15 cc. of urine) are pipetted into a 15 cc. centrifuge tube. (2) 1 cc. of the fluid (0.3 cc. of urine) is diluted with distilled water to 2 cc. in a 15 cc. centrifuge tube. (3) 2 cc. of the fluid (0.6 cc. of urine) are pipetted into a third 15 cc. centrifuge tube. All three tubes and one standard containing 2 cc. of the supernatant fluid are then carried through the general procedure at the same time. By comparing the amounts of precipitate after the addition of the uranyl zinc acetate reagent and

TABLE III

r

-

“;:Z- NO.

-

-

cc. w7.

0.12 119.2 0.12 322.5 0.20 172.3 0.20 83.7 0.60 64.7 0.60 66.0 0.60 60.7 0.60 61.1

Urine

Na in Na in sample standard

ml. mg.

0.1431 0.3148 0.3225 0.3148 0.3446 0.3148 0.0877 0.3148 0.3883 0.3935 0.3961 0.3935 0.3643 0.3935 0.3666 0.3935

-

I

._

-

ng. 0.2289 0.3509 0.3297 0.2076 0.3343 0.3388 0.3206 0.3219

-

I

-

-

va fount

ml.

0.2248 0.3500 0.3302 0.2018 0.3430 0.3392 0.3208 0.3208

-

1 I _-

1

RmCW- ‘=Y

wr cent

98.2 100.0 100.1 97.2

102.6 100.1 100.0 99.7

alcohol, the one approximating the standard is selected. If a duplicate is desired, the two closest to the standard are retained.

If the urine contains much protein, the latter is removed by adding sufficient solid trichloroacetic acid to make a 10 per cent solution. After thorough shaking, the specimen is filtered and the filtrate is neutralized to phenolphthalein with ammonium hydroxide and made up to such a volume that 10 cc. are equiva- lent to 3 cc. of urine. This is pipetted into a 15 cc. centrifuge tube containing the necessary amount of dry calcium hydroxide for the removal of phosphates.

The data in Table III show the recoveries when determinations were run in pairs, with one sample consisting of urine only, and the other of urine and a known quantity of standard sodium chloride.


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Tissue-Fresh beef muscle was used in all experiments. About 10 or 15 gm. of tissue are placed in a porcelain mortar and covered with 3 cc. of 20 per cent trichloroacetic acid. It is carefully macerated with the pestle until all protein has completely coagu- lated, after which 10 cc. of distilled water are added, and the maceration continued. The mortar is then tilted with the lip downward and the coagulat,ed mass pushed upward and pressed as dry as possible. With the solid mass held in place with the pestle, the extract is filtered into a 50 cc. beaker. The residue is further macerated with 2 cc. of trichloroacetic acid solution and 8 cc. of wairer. After the second extract is filtered into the beaker, 1 cc. of trichloroacetic acid is placed in the mortar, the pestle and mortar are rinsed with a few cc. of distilled water and maceration is continued. After this extract is filtered into the beaker, final extraction is made with a few cc. of distilled water. When the final extract has filtered through, the filter is washed three times with 2 cc. of distilled water. In another 50 cc. beaker are placed 2 cc. of standard sodium chloride stock solution (3.9345 mg. of Na per cc.) and 6 cc. of 20 per cent trichloroacetic acid solution, also sufficient distilled water to make a total volume of approximately that of the extract. The contents of the beakers are evaporated on a steam bath to approximately 15 cc. and neutralized to phenolphthalein with concentrated ammonium hydroxide. To each beaker is added 0.5 gm. of powdered calcium hydroxide. The contents are well stirred and digestion is continued for 4 hour, after which the beakers are cooled and allowed to stand at room temperature for 1 or more hours. The contents are filtered into 50 cc. volumetric flasks and the filters are washed as previously described. 2 cc. of both the sample and the standard are used in the precipitation of the triple acetate.

In working with larger quantities of tissue, e.g. 20 or 30 gm., proportionately larger quantities of trichloroacetic acid and calcium hydroxide are used, and the final volumes are made up to 100 cc.

Calculation-If the standard contains 0.3148 mg. of sodium, and the final volume of the extract from exactly 10 gm. of tissue is 50 cc., the following formula is employed.

R standard X 0.3148 X 100 R sample X 0.4

= mg. Na per 100 gm. tissue


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P. W. Salit 669

In five experiments, a different sample of beef muscle being used each time, pairs of determinations were run as follows: The tissue was divided into two unequal parts. To the smaller part a known quantity of standard sodium chloride solution was added in the mortar before maceration, and both samples were carried through the process in exactly the same way. The results are listed in Table IV.

Attempts were made to destroy the organic matter in tissue by dry ashing. Both nickel and vitreosol crucibles were employed, but the results were unsatisfactory. With new crucibles the results were too low, whereas after the crucibles were used the results were too high. The explanation suggested is that in new crucibles

TABLE IV

Tissue

s?-%?e Experi- ment

No.

1 1 2 1 3 1 4 1 5 1 5 2

Tissue, large

sample

gm.

0.3114 0.3119 0.2784 0 2879 0.7281 0.7281

Na, large sample

Na, 100 ?m.

tissue Na in

standard

ml. w. mg. ml. 0.1741 55.9 0.3148 0.2544 0.1760 56.5 0.3148 0.2809 0.1927 69.2 0.3148 0.3300 0 1828 63.5 0.3148 0.3403 0.4536 62.3 0.3148 0.6203 0.4718 64.8 0.3148 0.6294

T

Na found Rzy-

-- ml. per cent

0.2559 100.6 0.2810 100.0 0.3300 100.0 0.3472 102.0 0.6212 100.2 0.6212 98.7

some of the sodium fuses with the nickel or vitreosol and tends to accumulate in the crucibles.

Stool-Ashing of stools for sodium determinations is rejected for the above reasons. A procedure therefore was devised by which sodium is extracted with dilute alcohol. After the sample is thoroughly mixed in a beaker, it is placed in a short stemmed funnel. This is held over a tared 100 cc. lipless cylinder on a torsion balance, and, with the aid of a stirring rod, exactly 20 gm. of the material are delivered through the funnel into the cylinder. The cylinder is filled with 95 per cent alcohol to mark 90 cc., closed with a rubber stopper, and shaken until a more or less homo- geneous mass is obtained. After the addition of 1 cc. of concentrated ammonium hydroxide, the volume is made up with distilled water to 100 cc. In another similar cylinder, containing 1 cc.


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of concentrated ammonium hydroxide and 70 cc. of 95 per cent alcohol, are placed exactly 3 cc. of standard sodium chloride stock solution (3.9345 mg. of Na per cc.) and the volume made up to 100 cc. with distilled water. To each cylinder are added 2 gm. of powdered calcium hydroxide and the cylinders are stoppered and shaken. They are allowed to stand overnight at room temperature, the shaking of the sample being repeated at frequent intervals during the evening. The supernatant fluid of the sample is filtered into a small reagent bottle, then the entire contents of the cylinder are brought on the filter paper and covered with a watch- glass. Exactly 20 cc. of filtrate and standard are pipetted into 25 cc. volumetric flasks and evaporated to dryness in an electric oven at a temperature between 80-100’. Second portions of 20 cc. of each are pipetted into t,he same flasks and again evaporated to dryness. To each flask are added 2 cc. of perchloric acid and a drop of concentrated sulfuric acid; the contents are then digested over a low flame, an asbestos pad being used as a support. The digests are cooled, dissolved in 10 cc. of distilled water, and neutralized to phenophthalein with concentrated ammonium hydroxide. The volumes are made up to mark and 2 cc. of each are employed as outlined in ‘LGeneral procedure.”

Calculation-If a total of 40 cc. of the filtrate is evaporated, the

final quantity (2 cc.) of the sample will represent 20 x 40 x 2 = loo x 25

0.64 gm. of stool. The final quantity of standard will contain

(3 x 3.9345) x 40 x 2 = o 3777 mg. Na 100 X 25

Therefore, if the dilutions are the same for both the standard and sample, the final result, after the calorimetric readings, is

R standard X 0.3777 R sample X 0.64

= mg. Na per 1 gm. stool

Two different samples of stools were analyzed. Each consisted of a pair of determinations, one of which was carried out on the sample only (20 gm. of stool), the other on the sample plus a known quantity of standard (20 gm. of stool plus 4 cc. of standard sodium chloride stock solution). In the latter case only half the amount


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P. W. Salit 671

of filtrate specified above was evaporated for digestion. The results are given in Table V.

Ocular Humors--If the source is the human eye, from which slightly more than 0.2 cc. of aqueous humor can be obtained, exactly 0.2 cc. of the fluid is placed in a small test-tube and made up to 4 cc. with distilled water. It is well mixed with a stirring rod and 2 cc. are employed as outlined under “General procedure.” In working with animal eyes, larger quantities of fluid may be employed and diluted proportionately so that 2 cc. of the solution represent 0.1 cc. of the aqueous humor. Before diluting the vitreous humor in a similar fashion, the protein is precipitated by adding a few crystals of trichloroacetic acid to 10 cc. of vitreous humor and stirring until all is dissolved. After being filtered, it

TABLE V

Stools

-___ ~- ____~ 9”. ml. m7. ml. w. ml. per cent

1 0.44 65.1 ' 0.2264 0.3148 0.1725 0.1689 97.9 2 0.64 44.3 0 2835 0.3148 0 2678 0.2684 100.1

is treated like the aqueous humor. The standard is prepared as described for serum.

Results on ocular humors will be published later.

SUMMARY

A new triple acetate method for sodium is presented in which the sodium is precipitated with alcohol in a 15 cc. centrifuge tube, centrifuged, and washed with glacial acetic acid saturated with the triple salt. The sodium in the precipitate is estimated either gravimetrically or calorimetrically, depending on the amount of precipitate. The optimum quantity of sodium for the colori- metric procedure is approximately 0.3 mg. However, satisfactory results, with an experimental error of only 2 or 3 per cent, may be obtained with as little as 0.05 mg. of sodium.

The method is adapted, without resort to dry ashing, to sodium estimations in biological materials such as serum, whole blood, urine, tissue, stools, and ocular humors.


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My thanks are due to Dr. C. S. O’Brien, Head of the Depart- ment of Ophthalmology, for his valuable help and suggestions throughout the work.

BIBLIOGRAPHY

1. Streng, A,, 2. wissensch. Mikroskop., 3, 129 (1886). 2. Miholic, S. S., Bull. acad. SC. Zagreb., 16 (1920). 3. Blanchetibre, A., Bull. Sot. chim., 33, 807 (1923). 4. Barber, H. H., and Kolthoff, I. M., J. Am. Chem. Sot., 60,1625 (1928). 5. Barber, H. H., and Kolthoff, I. M., J. Am. Chem. Sot., 61,3233 (1929). 6. Butler, A. M., and Tuthill, E., J. Biol. Chem., 93, 171 (1931). 7. Caley, E. R., J. Am. Chem. Sot., 64, 432 (1932). 8. McCance, R. A., and Shipp, H. L., Biochem. J., 26,449 (1931).


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Peter Waldemar SalitBIOLOGICAL MATERIALS

FOR SODIUM DETERMINATIONS IN A NEW TRIPLE ACETATE METHOD

1932, 96:659-672.J. Biol. Chem.

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http://www.jbc.org/content/96/3/659.citation.full.html#ref-list-1

http://www.jbc.org/

a new triple acetate method for sodium de

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