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

21 Pages·2002·0.85 MB·English

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............................................... . 505
II. The method of measuring the surface tension.. . . . . . . . . 506
III. The effect of bile salts upon the surface tension. . . . . . 507
IV. Variation in the surface tension of normal urine.. . . 510
V. Urinary constituents not affecting the surface tension.. . 511
VI. The effect of peptones upon the surface tension of urine. . 512
VII. The effect of acetone bodies upon the surface tension of urine. . . 513
VIII. Benzoic acid and lactic acid.. . . . . . _ . . . . . . . 516
IX. The “reciprocal action” of mineral salts and bile salts in
lowering the surface tension.. . . . . . . . . . . . . . . . . . . . . . . 516
X. The effect of acidity upon the surface tension.. . . . . . . . . . . . . . . . 519
XI. Discussion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
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 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. Drops. S. T.
--
Distilled water ................................ 40.8 100.0
Na-Glyc. per cent in distilled water.
O.I........................... 59.8 68.2
0.05 .......................... 57.1 71.4
0.025 ......................... 55.0 74.1
0.0125 ........................ 52.6 77.5
0.00625 ....................... 40.5 82.4
0.003125 ...................... 46.3 88.1
0.0015625 ..................... 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.
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 .......................... I 40.8 100.0
Urine Sample I.
Diluted .............................. I 43.1 95.6
Urine + Nn-Glyc. gm. per 100 cc.
O.I........ ................ 65.7 62.7
0.05.. ...................... 63.0 65.4
0.025 ...................... 59.8 68.9
0.0125 ..................... 56.0 73.6
0.00625. ................... 52 :4 78.6
0.003125 ................... 49.0 84.1
0.0015625. ................. 46.0 89.6
0.00078125 ................. 44.4 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 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.
G. D. Allen 509
Surface tension in per cent.
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 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.
-r
As collected. )iluted to sp. gr. l.DlO
Date. Time. -
Sp. gr. DP3PS. s. T. DKlps. s. T.
td --
(Distills I ws,zt,er) 1 .ooo 40.8 100.0 40.8 100.0
Feb. 16 On rising 1.021 46.0 90.6 42.8 96.3
L‘ “ Soon 1.023 46.8 89.2 42.7 96.5
Feb. 19 On rising 1.022 48.3 86.1 43.4 94.9
LL “ Noon 1.019 43.2 96.2 41.6 99.1
(I ‘I 6 p.m. 1.024 45.7 91.4 42.3 97.4
LL “ 10 p.m. 1.028 46.8 89.6 42.1 97.9
Feb. 20 3 p.m. 1.025 45.7 91.5 42.1 97.9
Feb. 24 On rising 1.026 48.2 86.8 43.5 94.8
ii “ 11 a.m. 1.011 44.4 92.9 43.8 94.1
ii “ 1 p.m. 1.021 45.4 91.8 42.6 96.7
i< “ 6 p.m. 1.0195 44.7 93.1 42.6 96.7
(L I‘ 10 p.m. 1.024 46.2 90.4 42.6 96.7
Feb. 25 On rising 1.019 44.6 93.2 42.5 97.0
C‘ “ Soon 1.0215 44.0 94.7 42.0 98.1
Mar. 1 On rising l.Oil 45.7 91.2 42.4 97.2
Mar. 9 24 hrs. 1.0225 43.1 95.6
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.
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. Drops. s. T.
Distilled water 1.000 40.8 100.0
Urea 5.0 gm. per 100 cc. 1.013 41.4 99,s
I‘ 2.5 ‘I ‘I “ ‘i 1.006 41.0 100.1
Uric acid Saturated solution 1.002 40.7 100.2
IL I‘ 1 gm. in2000 cc. + 50 cc.
s NaOH +solid NaOI
till acid dissolved =
0.0487 gm. acid per
100 cc. 1.002 40.8 100.”
Hippuric acid 0.04 gm. per 100 cc. 40.8 100.0
Oxalic acid 0,004 I( LL I‘ “ 40.7 100.2
I‘ I‘ 0.002 “ (I “ “ 40.8 100.0
Creatinine 0.1 it LL (L ‘I 41 .o 99.5
Creatine 0.2 [[ (( ‘I if 40.9 99.5
‘I 0.1 [‘ (i ‘( LL 40.7 100.2
Allantoin 0.2 “ it tc tt 40.9 99.s
“ 0.1 “ (( (i (‘ 40.9 99.5
Inosite 1.0 ‘I ‘L (L L‘ 40.8 100.0
Distilled water (Temperature 26°C.) 41.0 100.0
Leucine 1.0 gm. per 100 cc. 41.7 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.
G. D. Allen 513
TABLE V.
Showing the Surjace Tension of Solutions of Witte’s Peptone in Distilled
Water and in Urine.
- -
Solution. sp. gr. Drops. S. T.
Distilled water.. . . . . . . . . . . . . . . . . . . . . . . . 1.000 41.0 100.0
Witte’s peptone 0.1 gm. per 100 cc.. . . . 50.5 81.2
,I “ 0.05 Cc (‘ Lc “ . . . . . 49.0 83.7
SL ‘I 0.02 “ “ “ “ . .., 45.9 90.0
L‘ I‘ 0.005 “ I‘ “ “ . . . 41.6 98.6
Distilled water.. . . . . . . . . . . . . . . . . . . . . . 1.000 41.0 100.0
Urine. Diluted. . . . . . . . . . . . . . . . . . . . 1 .OlO 44.0 94.0
Urine + Witte’s peptone 0.1 gm. per 100 cc 51.3 80.6
0.05 “ I‘ i: (6
‘( + “ “ 50.1 82.6
I‘ + “ “ 0.02 ‘L <L L‘ (I 47.2 87.7
(‘ + “ Lc 0,005 C‘ “ I‘ I( 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
depressedt he 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.. . . . . . . . . . . . . 1 .ooo 40.9 100.0
Acetone 2.0 cc. in 100 cc.. . . . . . 0.9975 45.9 88.7
t<
1 .o I‘ it (( “ . . . . . . 0.998 43.9 92.9
I‘
0.5 I‘ (L it I( . . 0.999 42.6 95.9
,L 0.25” ” ” ” . 1.000 41.9 97.6
Urine . . . . . . .._......_...._..._.._ 1.020 45.7 91.3
“ + 2.0 cc. zhcetonc in 100 cc. 1.0155 47.6 87.2
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. sp. gr. Y. T.
_~
Distilled water ............................ 1.000 40.9 100.0
Gm. per 100 cc.
@-Oxybutyric acid 1 .O ...................... 52.0 78.5
it Ii 0.75.. ................... 51.0 80.0
tt “ 0.5.. .................... 49.5 81.9
I< Ii 0.25. .................... 47.8 85.4
c< ” 0.125.. .................. 46.0 88.7
(L
” 0.0625.. ................. 44.5 91.7
cc ” 0.03125.. ................ 43.2 94.7
(‘ ” 0.007125 ................. 41.6 98.1
Distilled water.. . 1.000 40.8 100 .o
Urine...................................... 1.020 45.7 91.3
“ + 1.0 gm. /I-oxybutyric acid per 100 cc. 1.022 55.3 75.3
1. ooo
Distilled water. . . . . 40.9 100.0
&Oxybutyric acid 0.634 pm. per 100 cc.
after being made neutral to methyl orange
and alizarin with NaOH.. . _ . . 47.4 86.1
&Oxybutyric acid 0.645 gm. per 100 cc.
after being made neutral to litmus. with
NnOH. . . . . 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