THE DETERMINATION BY MEANS OF THE

BY (From the Department (Received

OF THE SURFACE
DELWIN University

BILE SALTS IN URINE TENSION METHOD.

ALLEN. of Minnesota, 13, 1915.) Minneapolis.)

GEORGE

of Physiology, for

publication,
CONTENTS.

August

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction............................................... The method of measuring the surface tension.. . . . . . . The effect of bile salts upon the surface tension. . . . Variation in the surface tension of normal urine.. . Urinary constituents not affecting the surface tension.. The effect of peptones upon the surface tension of urine. The effect of acetone bodies upon the surface tension of urine. Benzoic acid and lactic acid.. .. . . . _ . . . . . of mineral salts and bile salts The reciprocal action lowering the surface tension.. . ... . . . .. . . . .. ... . .. The effect of acidity upon the surface tension.. .. .. ... .. ... . Discussion.. ... .... ... ... ... ... .. ... ... .. ... .. ... . ... . .
I. INTRODUCTION.

.
.. .. . . . .. .. in .. .. ..

505 506 507 510 511 512 513 516 516 519 522

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Bile salts occur in the urine in certain diseases, especially jaundice, but the small quantities which occur are difficult to detect by chemical tests. Hays 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 concentrations, 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 Hays, but still convenient, and if possible a quantitative method, would be very desirable.
505

506

Bile Salts in Urine

The surface tension of a solution may be measured very accurately 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 constituents 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.

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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 22C. 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 The beaker of solution was then held with the stern of the instrument. 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 connection 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 The procedure reached the lower mark on the stem of the stalagmometer. 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 X specific gravity of the solution. Number of drops of 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.

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 surface. 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.

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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 Water.

01 Sodium

Gl~cocholtrlcin

Distilled

Drops. S. T .

Solution.

-Distilled water Na-Glyc. ................................ per cent in distilled O.I........................... 0.05 .......................... 0.025 ......................... 0.0125 ........................ 0.00625 ....................... 0.003125 ...................... 0.0015625 ..................... 40.8 water. 59.8 57.1 55.0 52.6 40.5 46.3 42.5 68.2 71.4 74.1 77.5 82.4 88.1 96.0 100.0

of distilled water from 100 per cent to 68.2 per cent by the addition 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 Eeubcrgs, en und Korperfltissigkeiten van Der Ham sowie die tibrigcu AusschcidungMcnsch und Tier, Rcrliu, 1011, pt. ii, 1709.

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 explained 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.

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Showing

the Surface

Tension
Solution.

oj Solutions

of Sodium
/ Sp. gr.

Glycocholate
1 Drops.

in
1

Urine.
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 I

40.8 43.1 65.7 63.0 59.8 56.0 52 :4 49.0 46.0 44.4

100.0 95.6 62.7 65.4 68.9 73.6 78.6 84.1 89.6 92.8

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 horizontal at about 60 per cent,. Donnan and Donnan found that, 0.0664 gram of sodium glycocholate 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.

G. D. Allen
Surface tension in per cent.

509

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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.

510
IV. VARIATION IN

Bile Salts in Urine

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 samples to a standard specific gravity of 1.010. When t.his was done the variation was founcl to be reduced. Table III shows the surface tension of the urine of the same individual taken at differTABLE III.

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Showin,g fhe Surface ut Different Times, after Being Diluted

Date.

(Distills Feb. 16 L Feb. LL (I LL 19 I

-r td

Tension of Samples oJ Urine Jrom the Smre IndividlLtrl and Showing the SurJace !lension oJ th,e Same Sampks to a Standard Sp. Gr. OJ 1.010.

Time. Sp. gr. 1 .ooo

As collected. DP3PS.

)iluted

to sp. gr. l.DlO

s. T. 100.0 90.6 89.2 86.1 96.2 91.4 89.6 91.5 86.8 92.9 91.8 93.1 90.4 93.2 94.7 91.2

DKlps.

--

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

Feb. 20 Feb. 24 ii ii i< (L I Feb. 25 C Mar. Mar. 1 9

I ws,zt,er) On rising Soon On rising Noon 6 p.m. 10 p.m. 3 p.m. On rising 11 a.m. 1 p.m. 6 p.m. 10 p.m. On rising Soon On rising 24 hrs.

1.021 1.023 1.022 1.019 1.024 1.028 1.025 1.026 1.011 1.021 1.0195 1.024 1.019 1.0215 l.Oil 1.0225

40.8 46.0 46.8 48.3 43.2 45.7 46.8 45.7 48.2 44.4 45.4 44.7 46.2 44.6 44.0 45.7

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

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 standard 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 IfIl~ SURFACE TENSION.

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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. Inorganic 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 concentrations 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 crcntine, oxalic acid, allantoin, inositc, and lcucine are also without effect. upon the surface tension of disOilled water in the conccntrat.ions indicated in Table IV.

512
Showing the Surface

Bile Salts in Urine

TABLE IV.

Tension

of Solutions in Distilled
Concentration.

of Various Water. sp. gr. 1.000 1.013 1.006 1.002 50 cc. NaOI = per 1.002 cc.

Urinary

Constifuevf.s

Drops.

Substmce.

s. T. 100.0 99,s 100.1 100.2

Distilled Urea I Uric IL acid I

water 5.0 gm. 2.5 I per 100 cc. I i

Hippuric acid Oxalic acid I I Creatinine Creatine I Allantoin Inosite Distilled Leucine water

Saturated solution 1 gm. in2000 cc. + s NaOH +solid till acid dissolved 0.0487 gm. acid 100 cc. 0.04 gm. per 100 0,004 I( LL I 0.002 0.1 0.2 0.1 0.2 0.1 1.0 it [[ [ I (I LL (( (i it (( L

40.8 41.4 41.0 40.7

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(L I I ( tc if LL tt

(i ( (L L

40.8 40.8 40.7 40.8 41 .o 40.9 40.7 40.9 40.9 40.8 41.0 41.7

100. 100.0 100.2 100.0 99.5 99.5 100.2 99.s 99.5 100.0 100.0 98.3

(Temperature 1.0 gm. per

26C.) 100 cc.

THE SURFACE

VI.

THE

EFFECT

OF

PEPTONES

UPON URINE.

TENSION

OF

The only other urinary constituents which we have found described as materially affecting the surface tension of urine are peptones. Lyon-Caen found that Wittes 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 appreciable error in attempting to measure the amount of bile salts present by the surface tension method.

G. D. Allen
TABLE V.

513
Peptone in Distilled

Showing

the Surjace

Tension

of Solutions
Water and in

of Wittes
Urine. sp. gr. 1.000

Drops.

S. T .

Solution.

Distilled water.. Wittes peptone ,I SL I L I Distilled Urine. Urine ( I ( water.. Diluted. Wittes

. ... ... ... .. .. .. .. . . ... 0.1 gm. per 100 cc.. . .. 0.05 Cc ( Lc . . . . . 0.02 . .., I .. . 0.005 . .. ... .. .. .... .. .. . . . ... ... .. .. ... .. . . . . peptone 0.1 gm. per 100 cc 0.05 I i: (6 0.02 L <L L (I 0,005 C I I( Lc

41.0 50.5 49.0 45.9 41.6 41.0 44.0 51.3 50.1 47.2 44.9

100.0 81.2 83.7 90.0 98.6 100.0 94.0 80.6 82.6 87.7 92.2

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1.000 1 .OlO

+ + + +

THE

VII.

THE

EFFECT

OF

ACETONE OF

BODIES URINE.

UPON

SURFACE

TENSION

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 depressedthe surface tension from 91.3 per cent t,o 87.2 per cent,
TABLE VI.

Showing

the Surface

Tension

of Solutions in Urine.

of Acetone

in Distilled

Water

and

Solution.

sp. gr.

Drops.

S. T .

Distilled Acetone t< I ,L Urine

water.. ... ... ... . 2.0 cc. in 100 cc.. . ... 1 .o I it (( . ... .. 0.5 I (L it I( . . 0.25 . . . . . . .._......_...._..._.._ + 2.0 cc. zhcetonc

. . .

1 .ooo 0.9975 0.998 0.999 1.000 1.020 1.0155

40.9 45.9 43.9 42.6 41.9 45.7 47.6

100.0 88.7 92.9 95.9 97.6 91.3 87.2

in 100 cc.

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

!lension

of Solutions Water and in

of @Oxybutyric Urine.
sp. gr.

Acid

in Distilled

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Solution.

Y. T . _~

Distilled @-Oxybutyric it tt I< c< (L cc (

water ............................ Gm. per 100 cc. acid 1 .O ...................... Ii 0.75.. ................... 0.5.. .................... Ii 0.25. .................... 0.125.. .................. 0.0625.. ................. 0.03125.. ................ 0.007125 .................

1.000

40.9 52.0 51.0 49.5 47.8 46.0 44.5 43.2 41.6

100.0 78.5 80.0 81.9 85.4 88.7 91.7 94.7 98.1 100 .o 91.3 75.3 100.0

Distilled water.. . Urine...................................... + 1.0 gm. /I-oxybutyric Distilled water. . .. &Oxybutyric acid 0.634 after being made neutral and alizarin with NaOH.. &Oxybutyric acid 0.645 after being made neutral NnOH. . .. .

acid per

100 cc. .

1.000 1.020 1.022

40.8 45.7 55.3 40.9

1.ooo

pm.

per

to methyl ._ . . gm. per 100 cc. to litmus. with

100 cc. orange 47.4 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

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 tension 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 acetonuria 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.

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Showing

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

sp. gr. 1.000

DropY.

S. T.

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 ( ( _. ..

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.

516
VIII.

Bile Salts in Urine

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 SALTS IN ACTION LOWERING THE OF MINERllL SURFACE SALTS TENSION. .4ND BILE

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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 tcn20 d ium salts were found more active in sion of distilled water. i 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 distilled water had a surface tension of 80.2 per cent of that of distilled 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 surface 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 glycocholate in distilled water, and have verified the fact t,hat a minimum surface tension is reached when the sodium chloride content 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, OBillard ,I., and

Jour. j. prakt. Chem., 1585. xxxi,

Dieulafh, Corn@. rend. Sot.

214. de biol., 1902, liv,

405.

G. D. Allen
TABLE IX.

517
Glycocholate in Distilled

Sh,owing

the Surface

Tension Water
Solution.

of Solutions and in Salt

of Sodium Solutions.

sp. gr. . 1 .ooo 1.001 1.0025 1.0035 1.005 1 .oos 1 .Oll 1.0135 1.000 1.002 1.0045 1.006 1.008 1.011 1.014 1 .ooo 0.997 1.005

Drops.

9. T 100.0 69.8 63.2 62.2 61.4 60.3 60.5 60.3 90.8 89.4 89.4 88.9 88.4 88.4 88.5 100.0 88.6 88.7

Distilled water.. Na-Glyc. 0.1 per St L

cent

40.7 58.4 64.6 65.7 66.6 68.0 68.0 68.4 44.8 45.6 45.7 46.0 46.4 46.5 46.6 40.9 46.0 46.3

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Na-Glyc. < (l 6 < ci Distilled Acetone

L L t<. < C

0.003125 per cent + 0.2 gm. NaCl I + 0.4 *i +o,fi Ii iL + 0.8 it + 1.2 it *i + 1.6 t<

per 100 cc. I Li L C it I i II C ci CL <c ii _, t< L c*

water.. 2 .O cc. per 100 cc. distilled CL CL I 1 .O per solution.................................

water cent NaCl

TABLE X.

Showing

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

in Distilled

sp. gr.

Drops.

s. T.

Distilled water.. Na- Glyc. 0.1 per I + LL ,f I + c< IL tt + :i (I ii +

.c i :c it f C C i i< L L< + C + (L + ii <I+ + +

. cent 0.2 0.4 0.6

.. . . . gm. Na#Od C C L Li
(I L CL it C L (I iL LL (L

per 100 cc. LL CL I L

ic L iI CL i< LC L I CL ! 6 L IL tc C L

0.8
1.0 1.2 1.4 1.6 2.0 10.0

1.000 1.001 1.0035 1.005 1.0065 1.008 1.009 1.0105 1.012 1.013 1.016 1.065

40.8 61.4 65.6 66.3 67.3 67.7 68.1 68.8 69.6 70.6 71.2 76.3

100.0 66.5 62.3 61.3 61.0 60.7 60.4 59.9 59.3 58.5 58.2 56.9

518

Bile Salts in Urine

TABLE XI.

Showing

the E$ect

upon

Surface

Tension

of Urine I .. . .. .. 100 cc.

n.f Adding sp. gr. 1.000 1.010 1.013 1.000 1.025 1.0285 1.0315 1.0345 1.0385

Sodium
lhpe. __-

Chloride
s. 1. __-

Solution.

Distilled water.. . Urine Sample I. . . . I I + 0.5 gm. NaCl Distilled water.. Urine Sample 6 I I C < , L . .I..

per

--

40.9 43.4 44.0 40.8 46.6 47.1 47.4 47.7 48.0 40.6 42.1 42.5 42.6 43.5 40.6 42.3 56.3

100.0 94.4 94.1 100.0 89.7 89.1 88.6 88.4 88.2 100.0 97.4 96.8 96.9 95.4 100.0 98.7 72.8

II.. < I

. . + 0.5 gm. NaCl + 1.0 < ti + 1.5 it L. +2,0 L .

per 100 cc. C < < L (I ( ( I( (L . gm. per gm. per gm. per . NaCl 100 cc. NaCl 100 cc. NaCl 100 cc. .

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-__

Distilled water.. .. Urine Sample II. Diluted.. I < ,c C i< L (L (L ( ic i:

.. + 0.5 + 1.0 +2.0

1.000 1.010 1.0135 1.0175 1.024 1.000 1.010

-.-

Distilled water.. ... . . . .. . . Urine Sample III. Diluted . . . I + 0.01 Glyc. I I + 0.01 Glyc. + 1.0 I

... . .... .

gm. Xaper 100 cc. gm. Naper 100 cc. gm. NaCl per 100 cc. + 0.01 gm. NaGlyc. per 100 cc. + 2.0 gm. NaCl per 100 cc.

1 .Ola5

57.8

71.5

1.025

59.0

70.5

figure until it reached 60.3 per cent for 0.8 per cent sodium chloride. 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.

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 lowering 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 possible 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.

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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 glycocholate, but when 0.01 per cent glycocholate is already present the addition of 0.003 per cent more of glycocholate causes a lowering of the tension of only 2 per cent, and when 0.1 per cent glycocholate is present a difference of 0.003 per cent more or less For this hardly causes an appreciable difference in the tension. reason the curves in Figure 1 based on observations upon four

520
Showing

Bile Salts in Urine

TAI(LE XII.

the Range of Surface Tension in Acid and in Alkaline

Solution.

of Solutions Samples

of Sodium

Glycocholate

of Urine.
sp. gr. 1.000 1.012 1.010
Drops. S. T .

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. NaGlyc. per 100 cc. Distilled water.. Urine Sample III. I cc < I( I[ C Ii L (6 .. . . . . . . (Reaction acid.). . . . Diluted.. + 0.1 gm. NaGlyc. per 100 cc. (cloudy) Diluted + 0.02 gm. NaGlyc. per 100 cc. (clear) Diluted + 0.005 gm. NaGlyc. per 100 cc.

40.8 42.4 42.3 60.7 57.3 53.0 47.0

100 .o 97.4 97.5 67.9

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71.9 77.7 87.7 100.0 83.9 91.9 60.8 68.0 77.7 100.0 90.9 95.1 65.8 70.5 77.3 81.0 86.0

1.000 1.028 1.010

40.8 50.0 44.8 67.7 60.6 53.0

Distilled water.. Urine Sample IV. I 1 I I, IL i I , C (

. . . .. . . . . . . . . . .. (Reaction alkaline.). Diluted.. . . +O.lgm.Na-Glyc. per 100 cc. i< I + 0.05 gm. NaGlyc. per 100 cc. 1 + 0.02 gm. NaGlyc. per 100 cc. ( + 0.01 gm. NaGlyc. per 100 cc. IL . + 0.005 gm. NaGlyc. per 100 cc.

1 .ooo 1.015 1.010

40.7 45.4 43.2 62.5 58.3 53.2 50.7 47.8

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

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.

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Showing

the E$ect

upon

the Surface

Tension Alkali.

of Urine

oj Adding

Acid

and

Solution.

/ Sp. gr. / Drops.

S. T.

Distilled Urine. LC l( LL ii ( t< CL

water.. Diluted.. C LL L LL tt LC IL <

. ... .. .. . . . . . . . . . . . . . . . + 1 drop concentrat,ed HCl per 100 cc. + 2drops + 3 I Ii + 4 (I (L IL I ( it Ii +5 ( + 7 Lt I( CL ti ii (I + 10 + 10 (lt Ii hrs. l later). .. . t< . I ic . Ii ic ( .

1.000 I .OlO 1.011 1.011 1.011 1.011 1.011 1,011 1.0115 1.0115 1 .ooo 1.010 1.010 1.011 1.011 1 .Oll

40.7 43.4 44.4 46.0 47.2 48.4 49.G 51.3 50.6 50.4 40.9 43.8 43.2 43.6 44.1 44.0

100.0 94.G 92.6 89.4 87.1 85.0 82.9 80.2 81.3 81.8 100.0 94.2 95.5 94.8 93.7 93.9

Distilled IJrine. I( I( C l

water.. Diluted. CL < 6

... . ... .. . (Another portion of same above.) . . . . . . .. . + 1 drop concentrated NaOH +2drops i( it +3 (L Lc $5 I G tL

... . sample as . . . . . per 100 cc Cl I i( Ii LL < Ii (L

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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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 indicate 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 produced 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 normal and icteric urines varies considerably with the reaction, being lower when the acidity is greater.
XI. DISCUSSION.

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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 Upon the basis of the due to the varying acidity of the samples. 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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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.

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Showing the Amounts Surface Tension 1.010.

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

05 Sodium Glycocholate in a Sample of Urine

Minimum cholate

Required to Give Any Observed Diluted to a Standard Sp. Gr. oj

to

of sodium glycowhich may give observed S. T . (Sample is acid.) Diluted earnpIe.

Maximum cholate

of sodium glycowhich may give observed S. T . (Sample ia alkaline.) Diluted gm. pa sample. 100 cc.

Per cent of that of distilled water.

gm. per 100 cc.

95 90 85 80 75 70 65 60

0 0 0.001 0.003 0.007 0.015 0.033 0.1+

0.001 0.004 0.008 0.015 0.03 0.065 0.1+ 0.1+

The taurocholate lowers the surface tension just a little tension. 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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Bile Salts in Urine

phenomenon of surface tension, and it gives no sat,isfactory objecThe stalagmometer does give a numerical t,ive numerical result. 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 1he amount of bile in the urine may be found of clinical value.

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