THE DETERMINATION OF THE BILE SALTS IN URINE BY MEANS OF THE SURFACE TENSION METHOD.

BY GEORGE DELWIN ALLEN.

(From the Department of Physiology, University of Minnesota, Minneapolis.)

(Received for publication, August 13, 1915.)

CONTENTS.

I. Introduction............................................... II. The method of measuring the surface tension.. . . . . . .

III. The effect of bile salts upon the surface tension. . . . IV. Variation in the surface tension of normal urine.. .

V. Urinary constituents not affecting the surface tension.. VI. The effect of peptones upon the surface tension of urine.

VII. The effect of acetone bodies upon the surface tension of urine. VIII. Benzoic acid and lactic acid.. . . . . . _ . . . . .

IX. The “reciprocal action” of mineral salts and bile salts lowering the surface tension.. . . . . . . . . . . . . . . . . . . . .

X. The effect of acidity upon the surface tension.. . . . . . . . . . . . . . XI. Discussion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 505 . . 506 . . 507 . 510 . 511 . 512 . . 513 . . 516 in . . 516 . . 519 . . 522

I. INTRODUCTION.

Bile salts occur in the urine in certain diseases, especially jaundice, but the small quantities which occur are difficult to detect by chemical tests. Hay’s method of testing by shaking flowers of sulphur upon the surface of the urine depends upon the unusual power which bile salts possess, even in small concen- trations, of lowering the surface tension of a solution. When the surface tension of the urine is lowered the powdered sulphur sinks to the bottom, and the lower the surface tension the more rapidly this takes place. The method is very unsatisfactory, however, because of the large variation in the surface tension of normal urine and because the interpretation of the test is so largely a matter of the judgment of the operator. Some test for bile salts more accurate than Hay’s, but still convenient, and if possible a quantitative method, would be very desirable.

505

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506 Bile Salts in Urine

The surface tension of a solution may be measured very accu- rately by the stalagmometric method. Donnan and Donnanl and Lyon-Caen2 have measured the surface tension of normal and pathological urines and of solutions of different urinary con- stituents by this method. The present st,udy was undertaken to determine the feasibility of est,imating the amount of bile salts present in pathological urines from measurements of the surface tension taken with a portable Traube stalagmometer. The problem was undertaken under the direction of Dr. J. F. McClendon, who has given valuable assistance at many points.

II. THE METHOD OF MEASURING THE SURFACE TENSION.

The surface tension of solutions rcportcd in this paper was measured by means of a portable Traube stalagmometer which drops disClIed mat,er at an average rate of 40.8 drops in about two minutes and a half at 22°C. Solutions were drawn up into the pipette with the assistance of a filter pump until the surface of the solution was above the upper mark on the stern of the instrument. The beaker of solution was then held with the surface of the solution in the bcakcr in contact with the lower end of the pipette. The solution was allowed to run out of the pipett,c until the upper meniscus of the liquid in the instrument rcachcd the upper mark on the stem. The beaker was then quickly lowered, breaking t,hc con- nection between the solution in the beaker and that in the pipette, and the number of drops flowing from the pipette was counted until the meniscus reached the lower mark on the stem of the stalagmometer. The procedure described enables one to estimate the fraction of the last drop very easily and at the same time to work rapidly.

The surface tension of a solution is computed in per cent of t,hat of distilled water according to the formula:.

Number of drops of distilled water Number of drops of solution

X specific gravity of the solution.

The results obtained by ‘these methods are in close agreement with those of Donnan and Donnan, who used more exact methods with a fixed pipette dropping about 201 drops of distilled water. As will be shown later, the determinations of Lyon-Caen were very high. This discrepancy appears to be due to t,he pipett.e which he used and describes as dropping 12Q drops of distilled

1 Donnan, TV. D., and I?. G., The Surface Tension of Urine in Health and Disease, Rrit. Med. Jour., 1905, pt. ii, 1636.

2 Lyon-Caen, L., Recherches .exp&iment,ales sur la tension supcrficielle des urines, Jour. de physiol. et de p&h. q&z., 1910, xii, 526.

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G. D. Allen 507

water in two to three minutes. This is nearly three t,imes the rat,e of dropping of t.hc Traube stalagmometer which we used, and is too great for accurate determinations. With too rapid dropping the substance which lowers the surface tension does not have time to accumulate in the surface layer enough to produce the maximum lowering. Bott.azzi3 states that in no case may more -than 20 drops a minut,e be permitted. In another respect also the pipette described by Lyon-Caen seems open to criticism; that is in the fact that it lacked a broad dropping sur- face. When a standardized Traube st.alagmometer is used it is our belief that measurements of the surface tension of urine can be made with sufficient accuracy for clinical purposes.

III. THE EFFECT OF BILE SALTS UPON THE SURFACE TENSION.

Bile salts possess the unusual property of lowering t,he surface tension of a solution very markedly even when present in small concentrations. Table I shows a lowering of the surface tension

TABLE I.

Showing the Xurjace Tension OJ Solutious 01 Sodium Gl~cocholtrlcin Distilled Water.

Solution.

Distilled water ................................ Na-Glyc. per cent in distilled water.

O.I........................... 0.05 .......................... 0.025 ......................... 0.0125 ........................ 0.00625 ....................... 0.003125 ...................... 0.0015625 .....................

-

--

-

Drops. S. T .

40.8 100.0

59.8 68.2 57.1 71.4 55.0 74.1 52.6 77.5 40.5 82.4 46.3 88.1 42.5 96.0

of distilled water from 100 per cent to 68.2 per cent by the addi- tion of 0.1 per cent sodium glycocholate.

Upon successive dilutions of this solut,ion the surface tension was gradually raised, but so small a concent,ration of the bile salt as 0.00156 per cent caused a lowering of 4 per cent,.

3 Bott.azzi, F., in Eeubcrg’s, Der Ham sowie die tibrigcu Ausschcidung- en und Korperfltissigkeiten van Mcnsch und Tier, Rcrliu, 1011, pt. ii, 1709.

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Bile Salts in Urine

When bile salts are added to normal urine their effect upon the surface tension is similar to that in distilled water. A specimen of normal urine diluted with distilled water to a specific gravity of 1.010 had a surface tension of 95.6 per cent. The reason for diluting with distilled wat.er to this specific gravity will be ex- plained later. When 0.1 gram of sodium glycocholate was added to 100 cc. of this diluted urine the surface tension was lowered to 62.7 per cent. Upon successive dilut.ions of this solution with the original urine (sp. gr. 1.010) the surface tension gradually increased. So small an amount of sodium glycocholate, however, as 0.00078125 gram in 100 cc., i.e., less t.han one part in 100,000 caused a lowering of nearly 3 per cent,.

TABLE II.

Showing the Surface Tension oj Solutions of Sodium Glycocholate in Urine.

Solution. / Sp. gr. 1 Drops. 1 S. T .

Distilled water .......................... Urine Sample I.

Diluted .............................. Urine + Nn-Glyc. gm. per 100 cc.

O.I........ ................ 0.05.. ...................... 0.025 ...................... 0.0125 ..................... 0.00625. ................... 0.003125 ................... 0.0015625. ................. 0.00078125 .................

I 40.8

I 43.1 95.6

65.7 62.7 63.0 65.4 59.8 68.9 56.0 73.6 52 :4 78.6 49.0 84.1 46.0 89.6 44.4 92.8

100.0

Figure 1 shows that increasing amounts of sodium glycocholate lowered the surface tension of this sample of urine (Sample I) at first very. rapidly and with further increases in concentration more slowly as the curve of surface tension approaches the hori- zontal at about 60 per cent,.

Donnan and Donnan found that, 0.0664 gram of sodium glyco- cholate in 100 cc. of urine lowered the surface tension from 91 per cent to 62.4 per cent. Lyon-Caen determined the surface tension of a 0.1 per cent solution of sodium glycocholate as 80.9 per cent. As explained above t,his high figure must be due to instrumental error.

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G. D. Allen

Surface tension in per cent.

509

FIG. 1. Curves showing the surface tension of solutions of sodiunl glycocholate in four samples of normal urine diluted to a standard specific gravity of 1.010.

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510 Bile Salts in Urine

IV. VARIATION IN THE SURFACE TENSION OF NORMAL URINE.

The surface t,ension of normal urine varies considerably even in the same individual. Donnan and Donnan found a variation in the same individual between 82.4 per cent and 93.6 per cent. They found the morning urine sometimes characterized by high density and low surface tension due to the greater ConcentraDion. Unusual exercise caused a fall in surface tension and a rise in density. In general a high specific gravity is associat,ed with a low surface t,ension. One cause of the variation in the surface tension of normal urine, t,herefore, is the varying dilution with water. If all samples of urine could be reduced to the average specific gravit,y, 1.020, t.his source of error could bc eliminated. Since t.his is not practicable we have attempted to dilute all sam- ples to a standard specific gravity of 1.010. When t.his was done the variation was founcl to be reduced. Table III shows t’he surface tension of the urine of the same individual taken at differ-

TABLE III.

Showin,g fhe Surface Tension of Samples oJ Urine Jrom the Smre IndividlLtrl ut Different Times, and Showing the SurJace !l’ension oJ th,e Same Sampks after Being Diluted to a Standard Sp. Gr. OJ 1.010.

Date.

(Distills Feb. 16

L‘ “

Feb. 19 LL “ (I ‘I LL “

Feb. 20 Feb. 24

ii “ ii “ i< “ (L I‘

Feb. 25 C‘ “

Mar. 1 Mar. 9

-r td

Time. Sp. gr.

I ws,zt,er) 1 .ooo

On rising 1.021 Soon 1.023 On rising 1.022 Noon 1.019

6 p.m. 1.024 10 p.m. 1.028 3 p.m. 1.025

On rising 1.026 11 a.m. 1.011

1 p.m. 1.021 6 p.m. 1.0195

10 p.m. 1.024 On rising 1.019 Soon 1.0215 On rising l.Oil 24 hrs. 1.0225

As collected. -

DP3PS. s. T.

40.8 100.0 46.0 90.6 46.8 89.2 48.3 86.1 43.2 96.2 45.7 91.4 46.8 89.6 45.7 91.5 48.2 86.8 44.4 92.9 45.4 91.8 44.7 93.1 46.2 90.4 44.6 93.2 44.0 94.7 45.7 91.2

)iluted to sp. gr. l.DlO

DKlps.

40.8 42.8 42.7 43.4 41.6 42.3 42.1 42.1 43.5 43.8 42.6 42.6 42.6 42.5 42.0 42.4 43.1

s. T. --

100.0 96.3 96.5 94.9 99.1 97.4 97.9 97.9 94.8 94.1 96.7 96.7 96.7 97.0 98.1 97.2 95.6

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G. D. Allen

cnt times of the day, and the surface tension of t,he samples when diluted to t,he standard specific gravity of 1.010.

The surface tension of samples at t,he standard specific gravity was above 94 per cent while the surface tension of t,he samples as collected ran down t,o 86.1 per cent. A sample was found from another individual which had a surface tension of 83.9 per cent and a specific gravity of 1.028. After dil&on to the stand- ard specific gravity the tension became 91.9 per cent. It seems to bc safe t,o conclude, therefore, t,hat if the surface tension of normal urine may run as low as 80 per cent, then the surface tension of samples of normal urine diluted to a standard specific gravity of 1.010 will lie above 90 per cent. In other words, the range of variation of normal urine is practically cut in half by this procedure. The measurements of the effects of bile salts and other substances upon the surface tension of urine have usually been made in this study upon samples of urine diluted t)o the standard specific gravity of 1.010.

V. URISARY CONSTITUENTS NOT AFFECTING ‘I’fIl~ SURFACE TENSION.

If the bile salts in urine are to be measured quant,itativcly 1)~ the surfncc tension method, it must be certain that the surface t#ension of the sample is not materially lowered or raised by other const,ituents. None of t,he normal comkituents of urine arc known to have any considerable effect upon the surface Qension. Inor- ganic salts increase the surface tension of distilled water, but, only very slightly. Donnan and Donnan found that urea, sodium urate, dextrose, acetone, and albumin (egg whit,e) had no effect, upon t.he surface Oension of urine, or only very lit& effect in con- centrations that are relatively high for urine. Lyon-Caen added creatininc, t,hiocyanates, hippuric acid, and biliary pigment.s to t.his list as not affect,ing the surface t,ension of distilled waler. We have confirmed these observations for urea, uric acid, hippuric acid, and creatinine in distilled wat#er, and have found that crc- ntine, oxalic acid, allantoin, inositc, and lcucine are also without effect. upon the surface tension of disOilled water in the conccn- trat.ions indicated in Table IV.

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512 Bile Salts in Urine

TABLE IV.

Showing the Surface Tension of Solutions of Various Urinary Constifuevf.s in Distilled Water.

Substmce. Concentration. sp. gr.

Distilled water Urea

I‘

Uric acid IL I‘

Hippuric acid Oxalic acid

I‘ I‘

Creatinine Creatine

‘I

Allantoin “

Inosite

0.04 gm. per 100 cc. 0,004 I( LL I‘ “

0.002 “ (I “ “ 0.1 it LL (L ‘I

0.2 [[ (( ‘I if 0.1 [‘ (i ‘( LL 0.2 “ it tc tt

0.1 “ (( (i (‘ 1.0 ‘I ‘L (L L‘

Distilled water (Temperature 26°C.) Leucine 1.0 gm. per 100 cc.

5.0 gm. per 100 cc. 2.5 ‘I ‘I “ ‘i

Saturated solution 1 gm. in2000 cc. + 50 cc.

s NaOH +solid NaOI till acid dissolved = 0.0487 gm. acid per 100 cc.

1.000 1.013 1.006 1.002

1.002

-

-

Drops.

40.8 41.4 41.0 40.7

40.8 40.8 40.7 40.8 41 .o 40.9 40.7 40.9 40.9 40.8

41.0 41.7

-

s. T.

100.0 99,s

100.1 100.2

100.” 100.0 100.2 100.0

99.5 99.5

100.2 99.s 99.5

100.0

100.0 98.3

VI. THE EFFECT OF PEPTONES UPON THE SURFACE TENSION OF

URINE.

The only other urinary constituents which we have found de- scribed as materially affecting the surface tension of urine are peptones. Lyon-Caen found that Witte’s peptone lowered the surface tension of distilled water to 88 per cent in a 0.1 per cent solution. We have found the effect to be still greater than is indicated by this figure. A 0.1 per cent solution in distilled water was found to have a surface tension of 81.2 per cent and 0.1 gram added to 100 cc. of urine lowered the tension from 94 per cent to 80.6 per cent. Peptonuria, therefore, may cause appre- ciable error in attempting to measure the amount of bile salts present by the surface tension method.

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G. D. Allen 513

TABLE V.

Showing the Surjace Tension of Solutions of Witte’s Peptone in Distilled Water and in Urine.

Solution.

Distilled water.. . . . . . . . . . . . . . . . . . . . . . . . Witte’s peptone 0.1 gm. per 100 cc.. . . .

,I “ 0.05 Cc (‘ Lc “ . . . . . SL ‘I 0.02 “ “ “ “ . .., L‘ I‘ 0.005 “ I‘ “ “ . . .

Distilled water.. . . . . . . . . . . . . . . . . . . . . . Urine. Diluted. . . . . . . . . . . . . . . . . . . . Urine + Witte’s peptone 0.1 gm. per 100 cc

‘( + “ “ 0.05 “ I‘ i: (6

I‘ + “ “ 0.02 ‘L <L L‘ (I

(‘ + “ Lc 0,005 C‘ “ I‘ I(

sp. gr.

1.000

1.000 1 .OlO

-

-

Drops. S. T .

41.0 100.0 50.5 81.2 49.0 83.7 45.9 90.0 41.6 98.6

41.0 100.0 44.0 94.0 51.3 80.6 50.1 82.6 47.2 87.7 44.9 92.2

-

VII. THE EFFECT OF ACETONE BODIES UPON THE SURFACE TENSION

OF URINE.

The acetone bodies, acetone, nceto-acetic acid, and p-oxybutyric acid, may occur in the urine in considerable amounts, especially in diabetes. Donnan and Donnan found that acetone added to urine had very little effect unless as much as 1 per cent by volume was added when the surface tension was depressed almost 3 per cent. We found that 2 per cent by volume of acetone in urine depressed the surface tension from 91.3 per cent t,o 87.2 per cent,

TABLE VI.

Showing the Surface Tension of Solutions of Acetone in Distilled Water and in Urine.

Solution. sp. gr. Drops. S. T .

Distilled water.. . . . . . . . . . . Acetone 2.0 cc. in 100 cc.. . . . .

t< 1 .o I‘ it (( “ . . . . . . I‘ 0.5 I‘ (L it I( . . ,L 0.25” ” ” ”

Urine . . . . . . .._......_...._..._.._ 1.020 45.7 91.3 “ + 2.0 cc. zhcetonc in 100 cc. 1.0155 47.6 87.2

. .

.

. 1 .ooo 0.9975 0.998 0.999 1.000

40.9 100.0 45.9 88.7 43.9 92.9 42.6 95.9 41.9 97.6

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514 Bile Salts in Urine

although 2 per cent of acetone in distilled water depressed the tension from 100 per cent to 88.7 per cent.

p-Oxybutyric acid is much more important in its action on the surface tension than acetone. A 1 per cent solution by weight in distilled water was found to have a surface tension of 78.5 per cent, and 1 gram in 100 cc. of urine depressed the surface tension from 91.3 per cent to 75.3 per cent.

TABLE VII.

Showing the Surfuce !l’ension of Solutions of @Oxybutyric Acid in Distilled Water and in Urine.

Solution.

Distilled water ............................ Gm. per 100 cc.

@-Oxybutyric acid 1 .O ...................... it Ii 0.75.. ................... tt “ 0.5.. .................... I< Ii 0.25. .................... c< ” 0.125.. .................. (L ” 0.0625.. ................. cc ” 0.03125.. ................ (‘ ” 0.007125 .................

Distilled water. . . . . &Oxybutyric acid 0.634 pm. per 100 cc.

after being made neutral to methyl orange and alizarin with NaOH.. . _ . .

&Oxybutyric acid 0.645 gm. per 100 cc. after being made neutral to litmus. with NnOH. . . . .

Distilled water.. . 1.000 Urine...................................... 1.020

“ + 1.0 gm. /I-oxybutyric acid per 100 cc. 1.022

1 .ooo

sp. gr.

40.9

Y. T .

_~

1.000 100.0

78.5 80.0 81.9 85.4 88.7 91.7 94.7 98.1

52.0 51.0 49.5 47.8 46.0 44.5 43.2 41.6

40.8 45.7 55.3

40.9

47.4

100 .o 91.3 75.3

100.0

86.1

47.6 85.7

When this acid was neutralized with sodium hydrate it was found to lose only about one-fourth of its effectiveness in lowering the surface tension of distilled water. When the 1 per cent @-oxybutyric acid solution in distilled water was neutralized to litmus with & sodium hydrate the resulting solution, which was equivalent to a 0.645 per cent solution of @-oxybutyric acid, had

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G. D. Allen 515

a surface tension of 85.7 per cent; while a distilled water solution of this concentration of the unneutralized acid has a surface ten- sion of 80.7 per cent as determined from a graph based upon Table VII.

In severe acetonuria the P-oxybutyric acid may be found in larger amounts than the acetone itself, and these observations indicate that such a condition would have a pronounced effect upon the surface tension of the urine. It would be important to determine also the effect of aceto-acetic acid upon the surface tension of urine for this substance would be found accompanying the oxybutyric acid, and it is also present in less severe acetonuria when the oxybutyric acid is not found. P-Oxybutyric acid, like the bile acids, is difficult to demonstrate in urine. Some simple test for it would be of great value in the study of glycosuria. Whether the surface tension method would be of value for this purpose is not certain. If aceto-acetic acid also lowers the tension very strongly, then these two bodies could not be distinguished by this means. It is evident, however, that conditions of ace- tonuria must be avoided in testing for bile salts by this method. According to von Noorden4 acetonuria can be eliminated, except in diabetes, by feeding carbohydrates.

TABLE VIII.

Showing the Surface Tension of Solutions of Lactic Acid and of Benzoic Acid in Distilled Water.

Solution.

Distilled water.. . . . Lacticacid1.0 gm.perlOOcc . . . . . . . . . .._

(6 c< 0.59 ‘I ‘I “ “ after being made neutral to litmus with NaOII.

Distilled water.. . . . . . . . . . Benzoic acid 0.2 gm. per 100 cc.. _.

‘6 *‘ 0.1 (I ‘[ “ (( 6‘ (: 0.05 (( cL ‘( ‘( . .

sp. gr.

1.000

DropY. S. T.

40.7 44.3

41.3

40.8 44.0 42.0 41.3

100 .o 92.1

98.8

100 .o 92.7 97.1 98.8

4 von Noorden, C., Clinical Treatises on the Pathology and Therapy of Disorders of Metabolism and Nutrition. Part VII. Diabetes Mellitus, New York, 1905.

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516 Bile Salts in Urine

VIII. BENZOIC ACID AND LACTIC ACID.

Lactic acid and benzoic acid have been found to have an effect upon the surface tension of distilled water, but a less effect than that of the peptones and the oxybutyric acid. These substances might become a minor source of error when present in considerable amounts.

IX. THE ‘LRECIPROCAL ACTION” OF MINERllL SALTS .4ND BILE

SALTS IN LOWERING THE SURFACE’ TENSION.

It has been shown by Traubej and by Billard and Dieulafe6 that while very dilute solutions of bile have a surface tension very much below that, of distilled wat,er, the tension is lowered st,ill further if a little mineral salt is added. This is true in spite of the fact that mineral salts themselves do not lower the surface tcn- sion of distilled water. i d 20 ium salts were found more active in this respect than those of pot,assium, and among t,he different, salts the chlorides were the most effective.

Lyon-Caen confirmed the results of 13illarcl and Diculaf6. llc found that a 0.1 per cent solution of sodium glycocholate in dis- tilled water had a surface tension of 80.2 per cent of that of dis- tilled water. The same concentration of glycocholate in 0.1 peg cent sodium chloride solution had a surface tension of 76.4 per cent. With increasing conccntrat,ions of sodium chloride the sur- face tension became successively lower until it reached 69.3 pci cent in an 0.8 per cent sodium chloride solution. This author made the further observation Ohat increase of sodium chloride above the concentration of 0.8 per cent, up to 1.5 per cent, at least, failed to cause greater lowering of the tension.

We have repeat,ed these experiments with sodium chloride in 0.1 per cent and 0.003125 per cent solutions of sodium glyco- cholate in distilled water, and have verified the fact t,hat a mini- mum surface tension is reached when the sodium chloride con- tent reaches 0.8 per cent,. The surface tension of a 0.1 per cent solution of glycocholate in diAlled water was found ho be 69.3 per cent. Increasing amounts of sodium chloride lowered t,his

5 Traube, ,I., Jour. j. prakt. Chem., 1585. xxxi, 214. OBillard and Dieulafh, Corn@. rend. Sot. de biol., 1902, liv, 405.

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G. D. Allen 517

TABLE IX.

Sh,owing the Surface Tension of Solutions of Sodium Glycocholate in Distilled Water and in Salt Solutions.

Solution. sp. gr. Drops. 9. T

Distilled water.. . Na-Glyc. 0.1 per cent

St L‘

1 .ooo 40.7 100.0 1.001 58.4 69.8 1.0025 64.6 63.2 1.0035 65.7 62.2 1.005 66.6 61.4 1 .oos 68.0 60.3 1 .Oll 68.0 60.5 1.0135 68.4 60.3

Na-Glyc. 0.003125 per cent ‘< ‘L “ + 0.2 gm. NaCl per 100 cc. (l “ ‘I + 0.4 “ “ ‘I “ Li ‘6 L‘ *i +o,fi Ii ‘L ‘C it I‘ <‘ t<. iL + 0.8 it ‘i II C‘ ci “ <‘ “ + 1.2 it CL <c “ ii _, ci ‘C *i + 1.6 t< t< ‘L c* “

Distilled water.. Acetone 2 .O cc. per 100 cc. distilled water

“ “ CL “ CL ‘I 1 .O per cent NaCl solution.................................

1.000 44.8 1.002 45.6 1.0045 45.7 1.006 46.0 1.008 46.4 1.011 46.5 1.014 46.6

1 .ooo 40.9 0.997 46.0

1.005 46.3

90.8 89.4 89.4 88.9 88.4 88.4 88.5

100.0 88.6

88.7 -

TABLE X.

Showing the Surface Tension of Solutions of Sodium Glycocholate in Distilled Water and in Sodium Sulphate Solutions.

Solution. sp. gr. Drops. s. T.

Distilled water.. . . . . . . . . . Na- Glyc. 0.1 per cent

I‘ “ ” + 0.2 gm. Na#Od per 100 cc. LL ,f ‘I + 0.4 ‘C ‘C LL “ CL c< IL tt + 0.6 L‘ Li I‘ L‘ “ :i (I ii + 0.8 (I ‘C “ LC 6‘

.c ‘C L< + 1.0 L‘ L‘ ic L‘ L‘

“ ‘C ‘C + 1.2 CL (I L‘ I‘ “

‘i i‘ (L + 1.4 “ “ “ CL IL

:c i< ii + 1.6 “ iL iI “ tc

it ‘L <I+ 2.0 “ LL CL !‘ C‘

f‘ “ “ + 10.0 it (L i< “ L‘

1.000 40.8 100.0 1.001 61.4 66.5 1.0035 65.6 62.3 1.005 66.3 61.3 1.0065 67.3 61.0 1.008 67.7 60.7 1.009 68.1 60.4 1.0105 68.8 59.9 1.012 69.6 59.3 1.013 70.6 58.5 1.016 71.2 58.2 1.065 76.3 56.9

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518 Bile Salts in Urine

TABLE XI.

Showing the E$ect upon Surface Tension of Urine n.f Adding Sodium Chloride .

Solution. I

Distilled water.. . . . . . . Urine Sample I. . . . . .

‘I I‘ “ + 0.5 gm. NaCl per 100 cc.

Distilled water.. . Urine Sample II.. . . .I..

6‘ ‘I ” + 0.5 gm. NaCl per 100 cc. ‘I C‘ <‘ + 1.0 <‘ ti C‘ <‘ ‘< “ <‘ ‘I + 1.5 it L. ‘L (I (‘ ,‘ L‘ “ +2,0 “ ‘L (‘ I( (L

Distilled water.. . . . . . . Urine Sample II. Diluted.. . .

‘I <‘ ,c C‘ + 0.5 gm. NaCl per 100 cc.

i< (L “ ic + 1.0 gm. NaCl per 100 cc.

‘L (L ‘( i: +2.0 gm. NaCl per 100 cc.

--

Distilled water.. . . . . . . . . . . . . . . . . . . . Urine Sample III. Diluted . . .

“ “ “ ‘I + 0.01 gm. Xa- Glyc. per 100 cc.

‘I ‘I “ “ + 0.01 gm. Na- Glyc. per 100 cc. + 1.0 gm. NaCl

per 100 cc. I‘ “ “ “ + 0.01 gm. Na-

Glyc. per 100 cc. + 2.0 gm. NaCl

per 100 cc.

sp. gr.

1.000 1.010 1.013

1.000 1.025 1.0285 1.0315 1.0345 1.0385

1.000 1.010

1.0135

1.0175

1.024

1.000 1.010

lhpe. __-

40.9 43.4 44.0

40.8 46.6 47.1 47.4 47.7 48.0

-__

40.6 42.1

s. ‘1‘. __-

100.0 94.4 94.1

100.0 89.7 89.1 88.6 88.4 88.2

100.0 97.4

42.5

42.6

43.5

40.6 42.3

56.3

96.8

96.9

95.4 -.-

100.0 98.7

72.8

-

1 .Ola5 57.8

1.025 59.0

71.5

70.5

figure until it reached 60.3 per cent for 0.8 per cent sodium chlo- ride. Further increase of sodium chloride up to 1.6 per cent caused no further lowering. Similarly the surface tension of a 0.003125 per cent solution of sodium glycocholate in distilled water was lowered from 90.8 per cent to 88.4 per cent by adding 0.8 per cent sodium chloride. Sodium sulphate was found to have a weaker “reciprocal action,” as Lyon-Caen also reports.

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G. D. Allen 519

Sodium chloride is the chief inorganic constituent in urine, and the fact that its “reciprocal action” reaches its maximum in an 0.8 per cent solution is suggestive. Urine normally contains a little more than this amount of salt, and adding a little more would not be expected to alter the surface tension. On the other hand diluting the samples to a standard specific gravity of 1.010 would reduce the concentration of this salt. Would it be possible to eliminate the small error from this source by adding an excess of sodium chloride? Table XI shows the effect upon the surface tension of urine of adding increasing amounts of sodium chloride.

The surface tension of normal urine to which no bile salts had been added was lowered by adding the salt. This was true both before and after diluting to 1.010 specific gravity. And the lower- ing continued after the 0.8 per cent solution of NaCl was exceeded. To the third sample of urine 0.01 per cent sodium glycocholatc was added which lowered the surface tension from 98.7 per cent’ to 72.8 per cent. Then an addition of 1.0 per cent NaCl lowered the tension 1.3 per cent more and a second addition of 1.0 per cent NaCl lowered the tension an additional 1.0 per cent. The rule, therefore, that a maximum lowering is reached in an 0.8 per cent solution does not hold true for urine, and it is not possi- ble to obtain uniform readings by adding an excess of NaCI. The error from varying concentrations of NaCl does not appra,r to be large, however.

X. THE EFFECT OF ACIDITY UPON THE SURFACE TENSION.

Samples of normal urine which have been diluted to a standard specific gravity of 1.010 have a surface tension above 90 per cent. It might be expected that the initial variation in different samples would tend to disappear with the addition of increasing amounts of bile salts. Thus a 10 per cent lowering of the surface tension of urine is caused by the addition of 0.003 per cent sodium glyco- cholate, but when 0.01 per cent glycocholate is already present the addition of 0.003 per cent more of glycocholate causes a lower- ing of the tension of only 2 per cent, and when 0.1 per cent glyco- cholate is present a difference of 0.003 per cent more or less hardly causes an appreciable difference in the tension. For this reason the curves in Figure 1 based on observations upon four

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520 Bile Salts in Urine

TAI(LE XII.

Showing the Range of Surface Tension of Solutions of Sodium Glycocholate in Acid and in Alkaline Samples of Urine.

Solution.

Distilled wat.er.. . . . . . . . . Urine Sample II. (Reaction not noted.).

“ I( “ Diluted.. . . . . . . <‘ 1‘ I: ‘( + 0.1 gm. Na-Glyc

per 100 cc. “ L( <L cc + 0.05 gm. Na-Glyc

per 100 cc. “ ‘L ,‘ ‘I + 0.02 gm. Na-Glyc

per 100 cc. ‘I iL (L ‘I + 0.005 gm. Na-

Glyc. per 100 cc.

Distilled water.. . . . . . . . . Urine Sample III. (Reaction acid.). . . .

I‘ cc “ Diluted.. ‘< I( I[ “ + 0.1 gm. Na-

Glyc. per 100 cc. (cloudy) ‘C L‘ ” Diluted + 0.02 gm. Na-

Glyc. per 100 cc. (clear) Ii (6 ” Diluted + 0.005 gm. Na-

Glyc. per 100 cc.

Distilled water.. . . . . . . . . . . . . . . . . Urine Sample IV. (Reaction alkaline.).

I‘ “ “ Diluted.. . . 1‘ ‘I “ “ +O.lgm.Na-Glyc.

per 100 cc. I, I‘ i<

“ ‘, ‘1

IL ‘C (‘

‘i (‘ IL

‘I + 0.05 gm. Na- Glyc. per 100 cc.

“ + 0.02 gm. Na- Glyc. per 100 cc.

“ + 0.01 gm. Na- Glyc. per 100 cc.

. “ + 0.005 gm. Na- Glyc. per 100 cc.

_

:.

sp. gr.

1.000 1.012 1.010

1.000 1.028 1.010

1 .ooo 1.015 1.010

Drops. S. T .

40.8 100 .o 42.4 97.4 42.3 97.5

60.7 67.9

57.3 71.9

53.0 77.7

47.0 87.7

40.8 100.0 50.0 83.9 44.8 91.9

67.7 60.8

60.6 68.0

53.0 77.7

40.7 100.0 45.4 90.9 43.2 95.1

62.5 65.8

58.3 70.5

53.2 77.3

50.7 81.0

47.8 86.0

different samples of urine (Tables I and XII) would be expected to approach each other toward their lower ends. As the figure indicates, a very different result was obtained. One sample of ‘urine to which 0.1 per cent glycocholate was added had a surface

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G. D. Allen 521

tension as high as 67.9 per cent and another sample as low as 60.8 per cent. The original samples (sp. gr. 1.010) in these two cases showed a difference in surface tension of 6.6 per cent.

The following observations called attention to the effect of the acidity of the urine upon its surface tension, and this factor seems to account for the discrepancy. Some samples of urine do not readily dissolve 0.1 gram of glycocholate in 100 cc. A sample of t,his sort had a specific gravity of 1.028 and a surface tension of

TABLE XIII.

Showing the E$ect upon the Surface Tension of Urine oj Adding Acid and Alkali.

Solution. / Sp. gr. / Drops. / S. T.

Distilled water.. . . . . . . . . . . . . . 1.000 40.7 Urine. Diluted.. . . . . . . . . . . . I .OlO 43.4

LC ‘C + 1 drop concentrat,ed HCl per 100 cc. 1.011 44.4 l( LL + 2drops ” “ ” “ ” 1.011 46.0 LL L‘ + 3 ” ” I‘ ” ” Ii 1.011 47.2 ii LL + 4 (I (L “ “ “ IL 1.011 48.4 “ tt +5 ‘( Ii I‘ “ ‘( it 1.011 49.G (‘ LC + 7 Lt I( CL ti ii (I 1,011 51.3 t< IL + 10 ” ” “ I‘ Ii ” 1.0115 50.6 CL ‘< + 10 Ii l‘ t< ic ic (‘

(lt hrs. later). . . . . . . 1.0115 50.4

100.0 94.G 92.6 89.4 87.1 85.0 82.9 80.2 81.3

81.8

Distilled water.. . . . . . . . . . . . . . . IJrine. Diluted. (Another portion of same sample as

above.) . . . . . . . . . . . . . . I( “ + 1 drop concentrated NaOH per 100 cc I( CL +2drops i( it Cl I‘ i( ‘C ‘<

+3 (L Lc “ “ Ii LL

l‘ ‘6 $5 I‘ ‘G tL ‘< Ii (L

1 .ooo

1.010 43.8 1.010 43.2 1.011 43.6 1.011 44.1 1 .Oll 44.0

40.9 100.0

94.2 95.5 94.8 93.7 93.9

83.9 per cent. When 0.1 gram of sodium glycocholate was added to 100 cc. of this urine a dense cloudy solution was obtained. The solution was then made clear by adding three drops of strong sodium hydrate, and it then had a surface tension of 64.3 per cent. When to a portion of this solution a drop of acid was added it became slightly cloudy and the tension dropped to 62.6 per cent. Another portion was diluted with the original urine to a 0.005 per cent glycocholate solution, when it had a tension of 75.1 per

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522 Bile Salts in Urine

cent. After adding a drop of acid t,o this solution the tension dropped to 71.7 per cent. The remainder of the original sample of this acid urine was diluted to a specific gravity of 1.010, when the surface tension became 91.9 per cent. Addit.ion of 0.1 gram of glycocholate to 100 cc. gave a partly cloudy solution having a tension of 60.8 per cent. This sample furnished the data for Sample III, Table XII and Figure 1. These observations indi- cate that a more acid reaction of icteric urine is accompanied by a lower surface tension.

The effect of acidity upon the surface tension of normal urine was then tested by adding increasing amount,s of hydrochloric acid to one portion and increasing amounts of sodium hydrate to another portion of the same sample of urine. The results are given in Table XIII, and show that a maximum difference of 15.3 per cent in the surface tension of a non-icteric urine was pro- duced by changing the acidity in this way.

The sample of urine which furnished the data for Curve III was known to be strongly acid. The reactions of Samples I and II had not been noted, but it was inferred that the latter was probably alkaline or weakly acid. To test this a fourth sample was obtained which was distinctly alkaline to litmus, and this sample (IV) gave a curve which approached that of Sample II.

We conclude, therefore, that the surface tension of both nor- mal and icteric urines varies considerably with the reaction, being lower when the acidity is greater.

XI. DISCUSSION.

When peptonuria and acetonuria are not present and when samples of urine are diluted to a standard specific gravity of 1.010 the chief remaining error in attempting to estimate the amounts of bile salts present by the surface tension method seems to be due to the varying acidity of the samples. Upon the basis of the data given above a table may be constructed showing the amounts of sodium glycocholate in urine diluted to a specific gravity of 1.010 which should be present in urine of any observed surface tension. Such a table is given in Table XIV which is based upon Figure 1. It indicates, for example, that if the surface tension

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G. D. Allen 523

is 85 per cent a minimum of 0.001 gram of sodium glycocholate per 100 cc. of the diluted sample may be present if the sample is acid, or a maximum of 0.008 gram may be present if the sample is alkaline.

Table XIV is based entirely upon data obtained wit,h sodium glycocholate. When bile is found in urine there is a mixture of glycocholate and taurocholat,e in unknown proportions, and, in many cases at least, biliary pigmems are also found. Lyon-Caen reports finding that biliary pigments do not affect the surface

TABLE XIV.

Showing the Amounts 05 Sodium Glycocholate Required to Give Any Observed Surface Tension in a Sample of Urine Diluted to a Standard Sp. Gr. oj 1.010.

S. T . of sample diluted to 1.010 sp. gr.

Per cent of that of distilled water.

95 90 85 80 75 70 65 60

Minimum of sodium glyco- Maximum of sodium glyco- cholate which may give cholate which may give

observed S. T . observed S. T . (Sample is acid.) (Sample ia alkaline.)

Diluted earnpIe. Diluted sample.

gm. per 100 cc. gm. pa 100 cc.

0 0.001 0 0.004 0.001 0.008 0.003 0.015 0.007 0.03 0.015 0.065 0.033 0.1+ 0.1+ 0.1+

tension. The taurocholate lowers the surface tension just a little less than the glycocholate does. Accordingly, the figures given in Table XIV are a little too low for a mixture of the two salts actually present in the urine. They represent the glycocholate equivalent of the mixture in its effect upon the surface tension.

While the results reported in this paper may be disappointing in not finding the stalagmometric measurement of t,he surface tension a very accurate means of determining the amount of bile salts in urine it must be remembered that no other simple test is as satisfactory. The Hay Oest also depends upon the same

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524 Bile Salts in Urine

phenomenon of surface tension, and it gives no sat,isfactory objec- t,ive numerical result. The stalagmometer does give a numerical result which is not dependent up6n the judgment of the operat,or. When the react,ion of the sample is taken into account together with the percentage surface tension, this method of estimating 1 he amount of bile in the urine may be found of clinical value.

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George Delwin AllenSURFACE TENSION METHOD

SALTS IN URINE BY MEANS OF THE THE DETERMINATION OF THE BILE

1915, 22:505-524.J. Biol. Chem. 

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