j. biol. chem.-1944-sobel-355-63

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

1/9

A CONVENIENT TITRIMETRIC ULTRAMICROMETHOD FORTHE ESTIMATION OF UREA AND KJELDAHL NITROGEN*

BY ALBERT E. SOBEL, A. MARGOT MAYER, AND SIDNEY P. GOTTFRIED(From the Division ofBiochemistry and the Pediatric Research Laboratory of Th e

Jewish Hospital of Brooklyn, Brooklyn, New York)(Received for publication, July 15, 1944)

The method originally described by Sobel, Yuska, and Cohen (1) for theestimation of urea and Kjeldahl nitrogen is modified for the application toultramicro quantities. For this purpose a simple micro aeration apparatusis employed having standard side arm test-tubes made of Pyrex glass.(This is illustrated schematically in Fig. 1.) For the inlet a l-hole stopperequipped with a glass tubing of small diameter reaching to the bottom isused. For the outlet the side arm is connected with a rubber tubing to theinlet of the receiving tube. The ammonia is aerated over, after treatmentwith alkali, to the receiving tube, which contains boric acid and the indi-cator mixture of Ma and Zuazaga (2) in slightly different proportions. Thetrapped ammonia is titrated with standard acid with a capillary microburette. The transfer of the ammonia is quantitatively completed in 20to 30 minutes. Forty to sixty simultaneous aerations may be set up withthe samepump. The material to be analyzed is digested (for Kjeldahl N)or treated with urease (for urea N) in the same ube from which the aerationis carried out and thus no transfer is required.The method was adapted to the estimation of urea in 0.1 ml. of bloodserum containing 5 to 100 y of urea N and for the estimation of total N,albumin N, and non-prot,ein N with 0.01 to 0.05 ml. of blood serum.Reagents--

1. Phosphate buffer pH 7.0. 15 gm. of sodium pyrophosphate, 2 gm. ofphosphoric acid (1.4 ml. of 85 per cent H3P04), 100 ml. of distilled water.2. Urease extract (3). 10 gm. of washed permutit are placed in a 1 literErlenmeyer flask. The permutit is washed with three 50 ml. portions of 2per cent acetic acid; then 100 ml. of 2 per cent acetic acid are added. 150ml. of 0.001 N sulfuric acid and 50 gm. of jack bean meal are added. Themixture is shaken for 15 minutes, 200 ml. of glycerol are added, and themixture again shaken for about 10 minutes and then put into the ice box.

* Partially presented before the Metropolitan Microchemical Society, November19, 1942, New York. Partially presented before the Microchemical Subgroup of thePhiladelphia Section of the American Chemical Society, Philadelphia, March 4,1944. Presented before the American Chemical Society, Division of Analytical andMicrochemistry, 107th meeting, April 3-6, 1944, Cleveland, Ohio.

355

byguest,onOctober15,2009

ww

w.jbc.org

Downloadedfrom
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

2/9

356 EST IMA TION OF UREA AND KJELDAHL NThe mixture is occasionally stirred by shaking and allowed to settle over-night in the refrigerator. The supernatant fluid is then removed andcentrifuged for 1 hour at 2000 R.P.M. The clear supernatant liquid is trans-ferred to a bottle and kept in the refrigerator. The extract so prepared isvery stable. One may also use commercially prepared urease tablets con-taining a buffer.

3. Indicator (2). 8 parts of 0.1 per cent bromocresol green in 95 per centalcohol and 1 part of 0.1 per cent methyl red in 95 per cent alcohol.

FIG. 1. Schematic diagram of micro aeration apparatus. 2 = trap for NH3 in air(2 per cent HaBOa); A = digestion tube, NH*+ OH-F NHI; B = receiving tube, 2per cent HaBOo + indicator.

4. 2 per cent boric acid with indicator. 20 gm. of boric acid are dilutedto 1 liter with distilled water. 100 parts of the 2 per cent boric acid aremixed with 1 part of the indicator.5. Urea nitrogen recovery solution. 2.1434 gm. of recrystallized ureaare dissolved in 1 liter of distilled water. 1 ml. contains 1 mg. of ureanitrogen.6. Urea nitrogen standard solution. 10 ml. of urea nitrogen recoverysolution are diluted to 100 ml. with distilled water. 1 ml. contains 0.1mg. of urea nitrogen.7. Ammonia-free caprylic alcohol saturated with thymol.8. Potassium carbonate. Half saturated solution, approximately 560gm. of the anhydrous salt per liter.9. Standardized 0.0714 N sulfuric acid.


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

3/9

SOBEL, MAPER, AND GO TTF RIED 35710. Digestion mixture. 500 ml. of distilled water, 30 gm. of potassium

sulfate, 5 gm. of copper sulfate, 480 ml. of concentrated sulfuric acid, 0.5gm. of selenium in 20 ml. of concentrated sulfuric acid.11. Trichloroacetic acid, analytical reagent grade, 5 per cent solutionin distilled water.

12. Sodium sulfate solution. 23 per cent solution in distilled water.This is kept in an incubator at 37.

13. Alkali solution. 12 parts of distilled water, 6 parts of sodium hy-droxide, and 1 part of sodium thiosulfate.

14. Ether, U. S. P.15. Standard and recovery solution for proteins. Stock solution:4.7186 gm. of ammonium sulfate, which has been dried overnight at 105,are dissolved in 500 ml. of distilled water. 1 ml. = 10 mg. Solution foruse: 1 ml. of the stock solution is diluted to 100 ml. with distilled water.1 ml. = 0.1 mg.

Apparatus-1. Pyrex test-tubeswith side arm (12 cm. in length and 1.5 cm. diameter).2. Glass tubing, 15 to 18 cm. in length, 4 mm. outside diameter, withdrawn out fine tip of about 1 to 1.5 mm. diameter.3. l-hole size 0 rubber stoppers.4. Rubber tubing, about 33 cm. in length, 8 mm. outside diameter, and

3.5 mm. inside diameter.5. Capillary micro burette (Rehberg, self-filling type).6. Hot-plate.7. Electric pump or water suction.

Procedure for Aeration and TitrationThe digestion tube of the aeration outfit containing 10 or 100 y of am-monia nitrogen and 1 ml. of distil led water is connected with a rubber tub-

ing to the receiving tube, which contains 1.5 ml. of the boric acid solutioncontaining the indicator. Then 0.5 ml. of the alkali solution for KjeldahlN is added to the digestion tube, the tubes immediately stoppered, and theaeration slowly started. After 20 to 30 minutes the ammonia is completelyaerated over and trapped in the boric acid; so the aeration is stopped byrapidly disconnecting the tubes from the pump. While the glass tubes,which are inserted into the receiving tubes, are being removed, they arecarefully washed down with a few drops of distilled water.

At the pH of the boric acid (4.2) the indicator is a faint pink, which turnsblue at a more alkaline pH, in this case caused by the ammonia. The boricacid solution is then titrated back to its original pH with the aid of a capil-lary micro burette. The tip of the burette is kept beneath the surface of


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

4/9

358 EST IMA TION OF UREA AND KJELDAHL Nthe liquid, which is stirred by means of a stream of air during this titration.A receiving tube containing the boric acid indicator mixture only serves formatching the color of the end-point.

Procedure for Micro-Kjeldahl Analysis0.2 ml. of digestion mixture is added to the sample to be analyzed, or to

0.1 ml. of standard ammonium sulfate solution in a control analysis, in adigestion tube and digested on a hot-plate. After it has cooled for a fewmoments, the walls of the test-tube are washed down with 1.0 ml. of dis-til led water. From here on the procedure of aeration and titration is con-tinued as described.

Table I shows the results obtained with 10 and 100 y of nitrogen in analy-ses of standard ammonium sulfate solution.

T ABLE IDetermination of Ammonia Nitrogen in 0.1 Ml. of Standard (NH&SO4 Solution

N foundN present 10.0~ I N present 100.0 y

Y Y Y Y10.0 10.2 98.6 99.410.0 10.0 98.0 100.010.5 9.8 99.4 99.89.8 10.0 99.6 99.010.0 9.8 100.0 100.0

Non-Protein Nitrogen-The protein of 0.1 ml. of serum is precipitatedwith 2.5 ml. of trichloroacetic acid and centrifuged for 10 minutes at 2000R.P.M. A 1.0 ml. aliquot is then digested with 0.2 ml. of digestion mixture.After it has cooled for a few moments, 1 ml. of distilled water is added andthe aeration and titration procedure continued as above.

Calculation-Titration X 2600 = mg. per cent of non-protein N.Total Protein (Total Nitrogen)-O.l ml. of serum is diluted to 1 ml. with

distilled water. 0.1 ml. of this mixture is digested with 0.2 ml. of digestionmixture on a hot-plate. The tubes are placed at an angle of about 45 andcare is taken that none of the liquid bumps out. The digestion is finishedwhen the liquid turns colorless or bluish green. It is allowed to cool for afew moments and 1 ml. of distilled water is added to wash down the walls ofthe test-tube. The aeration and titration are carried out exactly as de-scribed for known ammonium salts in the presenceof the digestion mixture.A reagent blank is run through simultaneously.


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

5/9

SOBEL, MAYER, AND GOTTFRIED 359Calculation-Total nitrogen, ml. of titration X 0.0714 X 14 = amount of N in

sample; ml. of titration X 10,000 = mg. per cent of Kjeldahl N.Total protein, (mg. per cent of total N minus mg. per cent of non-protein N) X6.25/1000 = gm. per cent of total protein.Albumin-The globulin of 0.2 ml. of diluted serum (0.1 in 1.0 ml.) is pre-cipitated with 0.3 ml. of sodium sulfate in a small test-tube (100 X 12 mm.)and shaken up with 1 ml. of ether for 1 to 2 minutes. The tubes are thencentrifuged for 10 minutes at 2000 R.P.M., 0.2 ml. aliquots of the aqueouslayer are digested with 0.2 ml. of digestion mixture, and the Kjeldahl nitro-

gen determined as described before.Calculation-Titration X 12,500 = mg. per cent of albumin N + non-protein N;(mg. per cent of albumin N + non-protein N minus mg. per cent of non-protein N) X6.25/1090 = gm. per cent of albumin.

TABLE IIDetermination of Nitrosen by Micro-Kjeldahl and Authors ~~tramicromethod in_ Blood SerumThe values are expressed in mg. per 100 ml. of serum.

Ir;on-proteinruKjeldahl Authors

33.4 33.133.6 33.334.3 35.134.5 34.837.6 38.039.2 , 39.0

Total NKjeldahl Authors

801 793835 825924 925946 9321024 1018

1079 1079

--I- Albumin NKjeldahl Authors

556 560583 587593 583608 604614 612716 706

Results-Table II shows the close agreement obtained when the authorsultramicromethod was compared with the micro-Kjeldahl method (2, 4).Procedure for Urea

0.1 ml. of serum, spinal fluid, or urine providing a sample with 10 to 200y of urea l?J, s added to the contents of a digestion tube which consists of 1drop of phosphate buffer, 1 drop of urease extract, and 3 drops of antifoamreagent. After an incubation period of 10 minutes 0.5 ml. of potassiumcarbonate is added, and the ammonia is aerated for 20 to 30 minutes.Urines are aerated a little longer becauseof the high values of urea. Other-wise, 0.1 ml. of urine may first be diluted to 1 ml. with distilled water, andthen 0.1 ml. of the mixture treated in the samemanner as serum. For thetitration a capillary micro burette is employed with 0.0714 N sulfuric acidto simplify the calculation.


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

6/9

360 EST IMA TION OF UREA AND KJELDAHL NCalculation-Mg. of urea N per 100 ml. of sample = (amount of urea N X lOO)/

(ml. of sample) = ml. of acid X 1000 when 0.0714 N acid is employed.Results-Table III shows the results obtained with 10, 100, and 200 y ofurea N. It may be noticed that the absolute error was larger with theordinary 1 ml. micro burette than with the capillary micro burette.

T ABLE IIIDetermination of Urea Nitrogen in 0.1 Ml. of Standard Urea Solution

Standard I&lo.; y urea N

Y10.010.010.010.210.010.010.010.210.09.8

Standard II,? 100 y urea N Standard 111,t 200 y urea Npresent presentY

100loo9610610010210010098100

Y200200202200200202202200198200

* Capillary micro burette.1 Ordinary 1 ml. micro burette.T ABLE IV

Comparison of Urea Nitrogen Valu es Determined on 1.0 Ml. of Serum with ThoseDetermined on 0.1 Ml. of Serum

The results are expressed in mg. per 100 ml.Micromethod Ultramicromethod Micromethod Ultramicromethod

17.2 17.2 56.4 56.516.6 16.6 18.0 17.818.2 18.2 10.0 10.228.4 28.5 14.2 14.514.4 14.3 21.2 21.6

Table IV shows he results obtained with 1 O ml. of serum by the methodof Sobel, Yuska, and Cohen (1) and those obtained by the authors methodwith 0.1 ml. of serum. The excellent agreement reached establishes thevalidity of the new method.In Table V results are shown which were obtained when known amountsof urea were added to 0.1 ml. of serum. The amounts of urea N recoveredwere very close o the theoretical amounts added, the error varying between0 and 0.4 per cent.


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

7/9

SOBEL, MAYER, AND GO TTFRIE D 361Table VI shows the average recovery of 100 urea nitrogen determinationswith standard solutions and with known amounts of urea added to 0.1 ml.

of serum. These values were obtained under daily routine conditions andnot by the investigators. The low standard deviation obtained under theseconditions indicates a high degree of precision even under routine conditions.

DISCUSSIONIt is important to remove impurities from newly obtained rubber stop-

pers and tubing. This is accomplished by boiling in a dilute solution ofTABLE V

Recovery of Urea Nitrogen Adde d to 0.1 Ml. of Serum100.0 y added to each sample.The values are expressed in micrograms of urea N.

Present in serum17.216.618.228.514.3

Recovered Present in serum Recovered190.0 56.5 99.8190.0 17.8 100.3100.2 10.2 100.0100.0 14.5 100.099.6 21.6 100.2

TABLE VIRecovery of Urea Nitrogen on 100 Routine Determ inations (Mean values)

The values are expressed in micrograms of urea N.Added Recovered Average Standarddeviation deviation

Standard solution.. . . . . . . 10.0 10.243 0.1445 0.19400.1 ml. urea solution added to 0.1 ml.serum....... ...... ..... ...... ..... ... 100.0 100.186 0.4432 , 0.6535

sodium hydroxide followed by washing with distilled water and finally with2 per cent boric acid.By using a wire basket on a hot-plate thirty Kjeldahl digestions may becarried out simultaneously with no bumping. In addition, the sameset-upcan be employed to distil off water in the serum or various serum filtratesas a preliminary step to digestion.The new antifoaming mixture, caprylic alcohol saturated with thymol,employed for the urea estimation probably works because the thymolalters the surface properties of the proteins in addition to the usual behaviorof caprylic alcohol in increasing interfacial tension. Caprylic alcohol alonedid not reduce foaming sufficiently to be useful. This was also true of a


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

8/9

362 ESTIMATION OF UREA AND KJELDA HL Nnumber of other higher alcohols tested. Castor oi l did prevent foaming butwas not as convenient to handle.

The indicator mixture of bromocresol green and methyl red gives a verysharp end-point, the change of the color being from blue to pink, passing acolorless state, which indicates the closeness of the end-point. Because ofthis sharp end-point this indicator is definitely preferable to Pattersonsindicator, with which the last change from blue to violet has to be watchedvery carefully. The ratio of 8 parts bromocresol green to 1 part of methylred was found to be more suitable for our purposes than the proportionsrecommended by Ma and Zuazaga (2).The aeration is started slowly and the air regulated with a clamp unti lthe air bubbles rise evenly throughout the system. The bubbles shouldpossibly not go up higher than half way to the side arm; otherwise thedanger exists that the liquid might be carried over to the next tube. In

T ABLE VIIInjkence of Diluting Serum 1~10 on Precipitation of Globulin with Sodium SulfateKjeld ahl N in filtrate, in mg. per 100 ml. of serum.

NazSO4 in final mixture13.8 per cent / 13.8 per cent 21.6 per cent

Diluted serum Undiluted seru m Undiluted seru mSamp le 1.. 718 895 712 2 660 825 658: 3 676 852 669

* Treated according to the technique described by the authors for album in deter-mination.order to obtain a small air bubble easy to regulate, the glass ubing is drawnout to a fine tip.The titration involves no special precautions. With practice a titrationwith a capillary micro burette takes no more time than with an ordinaryburette. Both the burette tip and the stirring tip are inserted into theliquid to be titrated. The stirring mechanism is attached to the microburette. (For our purposes an air stirring device was satisfactory.) Theair is purified by passing t through a glasscolumn containing calcium chlo-ride and soda lime. The tip of the stirring device is a glass tube of smalldiameter drawn out to a capillary opening. The speed of the air passingthrough the device into the solution is regulated with a clamp.The comparison of the solution with a pure boric acid indicator solutionis of help in titrating to the exact end-point. The volume of liquid in thematching color tube should be similar to the one of the solution to betitrated.


ww

w.jbc.org

Downloadedfrom

8/14/2019 J. Biol. Chem.-1944-Sobel-355-63

9/9

SOBEL, MAYER, AND GO TTFR IED 363

The reagent blank of urea nitrogen is negligible; the one of Kjeldahlnitrogen is of the order of 0.0002 to 0.0006 ml. of titration with 0.0714 Nacid.

The method of albumin estimation recommended requires further expla-nation. The final concentration of sodium sulfate in the procedure is 13.8per cent, at which concentration the precipitate is mostly euglobulin andthe filtrate pseudoglobulin I, pseudoglobulin II, albumin, and non-proteinnitrogen (5). However, it was found that when the serum was first diluted1: 10 with distilled water and 0.3 ml. of 23 per cent sodium sulfate wasadded to 0.2 ml. of the diluted serum the precipitate consisted of al l theglobulins. The results obtained compared well with the usual method, inwhich 3 ml. of 23 per cent sodium sulfate were added to 0.2 ml. of serum.The experimental proof for the validity of our method of albumin deter-mination is given in Table VII. When undiluted serum was added tosodium sulfate, higher results were obtained in the filtrate of a solutioncontaining 13.8 per cent sodium sulfate than with 21.6 per cent sodiumsulfate. This observation is in agreement with that of previous workers(5). However, the addition of diluted serum to 23 per cent sodium sulfateso as to produce a solution which is 13.8 per cent with respect to sodiumsulfate gave results similar to those usually obtained with 21.6 per centsodium sulfate.

Apparently the properties of globulins are changed when serum is dilutedwith distilled water, so that they precipitate more readily than they wouldotherwise. Though the globulins are known to be insoluble in water andalbumins are soluble, one observes a cloudiness but not a precipitate ondiluting serum, probably due to protective colloids present in the serum.

SUMMARYA simple titrimetric ultramicromethod employing aeration is described

for urea nitrogen, total protein, albumin, and non-protein nitrogen in bloodserum and other nitrogenous compounds. The method is rapid and a largenumber of determinations may be carried out simultaneously.

BIBLIOGRAPHY1. Sobel, A. E., Yuska, H., and Cohen, J., J. Biol. Chem., 118, 443 (1937).2. Ma, T. S., and Zuazaga, G., Ind. and Eng. Chem., Anal. Ed., 14,280 (1942).3. Raices, A. E., Rev. mbd.-quir. patol. femenina, 9, 598 (1937).4. Kingsley, G. R., J. Lab. and Clin. Med., 27, 840 (1942).5. Gutman, A. B., Moore, D. H., Gutman, E. B., McClellan, V., and Kabat, E. A.,J. Clin. Invest., 20, 765 (1941).


ww

w.jbc.org

Downloadedfrom

j. biol. chem.-1944-sobel-355-63

Documents