THE RELATION OF UREA TO THE MOVEMENT OF WATER IN - PNAS
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
THE RELATION OF UREA TO THE MOVEMENT OF WATER IN
LIVER TISSUE
EUGENE L. OPIE
THE ROCKEFELLER INSTITUTE
Communicated February 1, 1960
The purpose of this study has been to determine what conditions maintain the
high level of osmotic pressure within liver cells present immediately after their
removal from the body, to define the relation of water movement to this pressure,
and to learn if intracellular osmotic pressure and water movement are changed
when amino acids and related substances are transformed to produce urea.
Significant information concerning the activity of liver or of kidney cells as osmom-
eters is obtainable immediately after the tissue is removed from the body.' With
relation to a wide variety of electrolytes, liver cells acting as osmometers measure
the osmotic pressure of solutions in accord with the molecular weight, valence, and
ion-dissociation of each substance. Tables (Robinson and Stokes2) assembled from
freezing point depression, vapor tension, electrical conductivity, and other colliga-
tive properties of electrolytes have made it possible to define the molar concentra-
tion of electrolyte solutions with the same osmotic pressure. A solution of sodium
chloride isotonic for liver immediately after its removal from the body is approxi-
mately 0.34 molar and a similar solution for kidney, 0.23 molar. Water equilib-
rium is maintained by the tissue at these levels during 15 to 20 min. Electrolytes
as varied as potassium chloride, with monovalent base, magnesium chloride, with
bivalent base, and lanthanum chloride with trivalent base, are isotonic with liver or
kidney tissue in solutions that have the same osmotic pressure as that of sodium
chloride in solution isotonic with one or other of these tissues. The tissues, acting
as osmometers, measure osmotic pressure with approximate accuracy.
The plasma membrane of liver cells is evidently imperfectly semipermeable to
solutions of electrolytes and with their entrance into the cell permeability to both
electrolyte and water is increased. Maintenance of isotonicity in vitro is promoted
by immersion in media with electrolytes similar to those of blood plasma, by tem-
perature at 380 C and by an adequate supply of oxygen.5
Slices of liver tissue, weighing preferably from 60 to 90 mg, determined rapidly
by means of a torsion balance, have been immersed in the Krebs3 modification of
Ringer's solution much used for manometric measurement of oxygen consumption.
The solution with electrolyte concentration of 0.154 molar has been buffered with
potassium phosphate and treated with oxygen or buffered with sodium bicarbonate
and gassed with oxygen, 95 per cent, and carbon dioxide, 5 per cent. The medium
is hypotonic for liver tissue but when its molar concentration is increased two-fold
by addition of sodium chloride isotonicity may be maintained during one or two hr.4
Water movement measured in per cent of weight before immersion varies widely
with oxygen supply to the medium. With abundant oxygen under conditions that
promote its diffusion throughout the medium and into the tissue, water enters
rapidly during about one hr but later is lost and within 2 or 3 hr may be less than
that before immersion. With anoxia caused by diminished oxygen supply or by
substitution of nitrogen for oxygen, water intake during the first hr of immersion
477
Downloaded by guest on November 11, 2021478 BIOCHEMISTRY: E. L. OPIE PROC. N. A. S.
may not be significantly different from that with oxygen, but later water of the tissue
increases during the next two or three hr.5
The opinion that the osmotic activity of tissues is determined by their electrolyte
content, chiefly by potassium and sodium, has had wide acceptance but there is
much evidence to indicate that protein and substances concerned with metabolism
may have an important part in maintaining intracellular osmotic pressure. When
liver tissue is subjected to a temperature approximately 380, it undergoes autolysis
and if slices prepared from tissue that has been kept at this temperature are im-
TABLE 1
ISOTONICITY OF LIVER TIssUE INCUBATED AT 380C
Isotonic with solutions
of sodium chloride molar
Before immersion 0.37
After 30 min 0.48
After 60 min 0.51
After 90 min 0.81
Small blocks of liver tissue, all from the same animal, have been incubated at 380C for periods varying from 30
to 90 miin. Slices prepared from them have been immersed in graded solutions of sodium chloride during 10 min.
mersed in solutions of sodium chloride with concentration graded from 0.1 to 0.5
molar, the level of isotonicity will be found to have increased with the duration of
incubation (Table 1) so that after one and a half hr it may have reached 0.8 molar.
This change is best explained by the assumption that autolysis, with disintegration
of protein, has resulted in the formation of organic substances with relatively small
molecular weights. When liver slices are made from tissue that has been kept at
approximately 0WC, its level of isotonicity does not increase (Table 2).
TABLE 2
ISOTONICITY OF LIVER TISSUE KEPT AT 0.30C
Isotonicity of Isotonicity of liver
liver tissue At 0.30 tissue kept at 0.30C
Experiment molar min molar
1 0.37 120 0.38
2 0.36 125 0.39
3 0.38 145 0.35
4 0.27 153 0.20
Av 0.35 ... 0.34
Small blocks of liver tissue have been kept in broken ice and water (approximately 0.31C) during the time
indicated. Slices have been prepared and immersed in graded solutions of sodium chloride, 0.1 to 0.5 molar,
during 10 min.
The molar concentration of solutions of a wide variety of electrolytes isotonic with
liver tissue coincides with the osmotic pressure each maintains because the liver cell
is an approximately exact osmometerl but similar information concerning organic
nitrogenous substances is not readily available. Nevertheless, it is possible to
determine for some nitrogenous substances the molar concentration that is isotonic
with liver and to compare it with the concentration of sodium chloride similarly
isotonic with liver tissue immediately after its removal from the body (Table 3).
Wide variation in the osmotic pressure maintained by amino acids, urea, and
some nitrogenous substances related to them suggest the possibility that these
substances, intimately associated with protein metabolism, may have a part in deter-
mining intracellular osmotic pressure and movement of water.
The formation of urea by the action of arginase on arginine was described by
Kossel and Dakin.6 The reactions by which arginine, ornithine, and citrulline form
Downloaded by guest on November 11, 2021VOL. 46, 1960 BIOCHEMISTRY: E. L. OPIE 479
urea were defined by Krebs and Henseleit.3 These substances (Table 2) are iso-
tonic with liver cells in molar concentration approximately that of sodium chloride,
whereas urea and closely related substances which are readily diffusible are isotonic
with liver cells only when in highly concentrated solution. It has seemed probable
that protein, amino acids, especially arginine, together with ornithine and citrul-
line and doubtless other nitrogenous substances, contribute to the osmotic pressure
within liver cells. This relation suggests that intracellular pressure is perhaps di-
minished when urea is formed, and experiments have been made to determine if
water movement in liver slices is related to the formation of urea.
TABLE 3
MOLAR CONCENTRATION OF SOLUTIONS OF UREA, AMINO ACIDS, AND RELATED SUBSTANCES
- ISOTONIC WITH LIVER TISSUE
Molar
Sodium chloride 0.34
Arginine 0.26(3)
Arginine hydrochloride neutralized 0.24(1)
Ornithine hydrochloride neutralized 0.25(2)
Guanidine hydrochloride neutralized 0.29 (3)
Choline chloride 0.28(2)
Citrulline 0.47 (2)
Creatinine 0.49(2)
Glyeine 0.72(1)
Alanine 0.71 (?)
Thiourea 0.85(2)
Methyl urea 1.26(2)
Urea 1.56(3)
The number of determinations is shown by the figures in parenthesis. The hydrochlorides of arginine, ornithine,
and guanidine have been neutralized with choline hydroxide (pH 7).
The colorimetric method of Archibald7 has been used to measure urea formation.
Protein has been removed from homogenates of tissue slices and from immersion
media by heating to 1000C, in the presence of a sodium acetate buffer. Urea is
determined by the red color which appears when it is heated with alpha-isonitroso-
propiophenone.
Several procedures have been tested to determine in vitro the relation of urea
formation to oxygenation of the medium. Liver slices have been immersed in
Krebs-Ringer solution buffered with potassium phosphate and supplied with oxygen
(100 per cent) allowed to enter at the bottom of a large test tube (low bubbling) or
about 0.5 cm below the surface of the column of fluid in the tube (high bubbling).
With the less effective oxygenation of the high bubbling gas there has been a slight
increase of urea above that in the tissue before immersion, whereas with oxygen
bubbling into the lowermost part of the fluid and in contact with the tissue, urea,
after 3 hr of immersion, has been from 2 to 3 times that in the liver tissue immedi-
ately after its removal from the body. In these experiments water has been taken
into the tissue during the first hr of immersion but later, with continued oxygena-
tion, the increased water has usually been lost. With less opportunity for oxygen
to diffuse throughout the medium (high bubbling) water intake has increased con-
tinuously during several hours. Later experiments, made under the same condi-
tions but with liver slices in Krebs-Ringer fluid buffered with sodium bicarbonate
and exposed to oxygen 95 per cent and carbon dioxide 5 per cent, had similar results
but showed that more urea was formed in the presence of carbon dioxide than in
oxygen alone.
Downloaded by guest on November 11, 2021480 BIOCHEMISTRY: E. L. OPIE PRoc. N. A. S.
More constant results have been obtained when liver slices are immersed in fluid
with wide surface exposed to streaming gas and agitated by rhythmic shaking.
Flasks with flat bottoms (Erlenmeyer) and capacity of 90 cc have contained 25 cc
of fluid. Shaking has been at the rate of 130 per min.
The average urea content of liver slices in fourteen experiments (Table 4) has
been 33.7 mg per 100 gm. of tissue with variation from 14.9 to 50.1 mg. After 3 hr
of immersion, increase of urea has been approximately 3 to 6 times that of the tissue
before immersion, and after 4 hr as much as 13 times that of the control, the average
increase being 113.3 mg per 100 gm. of tissue and with variation from 61 to, 329 mg.
Urea leaves the slices and their urea content has diminished with the progress of
immersion (not shown in Table 4). In four experiments the average urea content of
the liver tissue has been 37.3 mg per 100 gm. and after 60 min of immersion has de-
creased to 11.2 mg. It has later remained little changed, being after 120 min, 10.4
mg, after 180 min, 8.1 mg and after 240 min, 9.6 mg.
Total increase of urea (in tissue slices and immersion fluid) has been accompanied
by some water intake during 60 min, but water has later been withdrawn from the
the liver slices and its diminution has been continuous.
The formation of urea in the presence of oxygen and carbon dioxide (Table 4) has
been compared with that which occurs with anoxia (Table 5) produced by the sub-
TABLE 4
UREA FORMATION AND WATER MOVEMENT IN LIVER SLICES WITH OXYGENATION
- ----Urea - .- - Water--
Before Before
immer- After After After After immer- After After After After
sion 60 min 120 min 180 min 240 min sion 60 min 120 min 180 min 240 min
Exp. mg mg mg mg mg % % % % %
1 38.2 79.0 .... 137.3 .... 100.0 99.8 .... 95.0 ....
2 14.9 67.0 80.7 102.1 .... 100.0 109.3 108.3 103.4
3 19.2 82.2 180.9 129.0 258.3 100.0 104.5 99.2 105.0 94.9
4 38.7 ... 87.6 97.3 .... 100.0 .... 97.6 99.3
5 36.4 ... 82.1 99.3 111.4 100.0 .... 96.3 85.0 85.3
6 26.9 61.3 .... 76.8 85.9 100.0 98.1 .... 88.5 86.0
7 34.9 89.9 .... 121.3 132.5 100.0 109.0 .... 101.2 104.7
8 31.7 68.5 78.9 90.2 100.0 100.0 97.5 94.7 97.1 83.1
9 34.8 78.5 94.2 110.6 .... 100.0 112.0 110.0 110.5 ....
10 26.9 62.9 75.0 89.5 .... 100.0 106.0 101.3 99.9 ....
11 32.8 86.2 119.0 134.2 136.8 100.0 107.1 99.7 98.3 102.8
12 45.3 58.5 116.5 143.5 .... 100.0 102.1 102.3 96.8 ....
13 50.1 ... 137.8 .... 186.9 100.0 .... 107.1 .... 94.9
14 40.5 ... 86.0 .... 164.5 100.0 .... 80.6 .... 82.6
Av33.7 73.4 103.5 110.9 147.0 100.0 104.5 99.7 98.8 93.0
Liver slices have been immersed in Krebs-Ringer solution with bicarbonate buffer in flasks (with 25 cc) gassed
with oxygen 95 per cent and carbon dioiide 5 per cent. Numbers are given to the experiments without relation
to the order in which they were made.
TABLE 5
UREA FORMATION AND WATER MOVEMENT IN LIVER SLICES WITH ANOXIA
Urea W ater
Before Before
immer- After After After After immer- After After After After
sion 60 min 120 min 180 min 240 min sion 60 min 120 min 180 min 240 min
Exp. mg mg mg mg mg % % % % %
6 26.9 37.4 ... 40.5 35.5 100 121.3 .... 105.8 113.8
15 39.4 46.9 62.2 47.7 49.4 100 122.3 129.9 129.1 129.0
13 50.1 ... 67.1 ... 65.4 100 .... 132.7 .... 130.6
14 40.5 ... 53.3 ... 54.8 100 .... 107.0 .... 112.8
AV39.22 42.15 60.86 44.1 51.27 ... 121.8 123.2 117.5 121.6
Liver slices have been immersed in Krebs-Ringer solution gassed with nitrogen 95 per cent and carbon dioxide
5 per cent, but otherwise under conditions the same as those of experiments recorded in Table 4. Experiments
with the same numbers as those of Table 4 were in each instance made with liver slices from the same animals.
Downloaded by guest on November 11, 2021VOL. 46, 1960 BIOCHEMISTRY: E. L. OPIE 481
stitution of nitrogen for oxygen but otherwise under the same conditions. Some
urea formation occurs when the medium is gassed with nitrogen and carbon dioxide
but it is considerably less than that with oxygen and carbon dioxide, the quantity
per 100 gm. of tissue increasing with oxygenation from 34.8 mg per 100 gm. of tissue
to 134 mg after 4 hr, whereas similar increase with anoxia in the presence of nitrogen
has been from 39.2 to 51.3 mg. With anoxia in the presence of nitrogen and carbon
dioxide, water increase has been approximately 20 per cent but with oxygenation,
after scant increase during one hr water has been lost by the slices.
A solution of the amino acid arginine (Table 3) is in water equilibrium with liver
slices when its molar concentration is somewhat less (0.25 molar) than that of a
sodium chloride solution (0.34 molar) isotonic with liver. Of two substances re-
lated to arginine and concerned in the formation of urea, one ornithine, is isotonic
with liver slices in solutions with molar concentrations the same as that of arginine
and the other, citrulline, in somewhat more concentrated solution. Substances with
the osmotic properties of arginine, ornithine, and citrulline accumulating in the cell
would draw water into it but in the presence of adequate oxygenation these sub-
stances form urea which, being rapidly diffusable, leaves the cell as soon as its con-
centration exceeds that of the surrounding medium. With anoxia, on the contrary,
nitrogenous products of metabolism may supplement the pressure maintained by
ions of potassium, sodium, and other electrolytes.
With the formation of urea from arginine and related substances in view, amino
acid nitrogen has been measured in order to determine its changes with oxygenation
and with anoxia. Amino acid nitrogen of immersion fluids with the homogenized
tissue slices has been measured by the method of Folin,8 comparing color changes
with sodium beta-naphthoquinonesulphonate with those of glycine used as a stand-
ard. The precautions recommended by Natelson9 have been followed. Protein
has been removed from homogenates of tissue slices and from immersion fluids by
precipitation with tungstic acid. Amino acid nitrogen has been measured in mg per
100 gm. of the immersed tissue slices. In some instances the slices have been ho-
mogenized and amino acid nitrogen of slices and of immersion fluid measured sepa-
rately.
TABLE 6
AMINO ACID NITROGEN IN LIVER SLICES WITH OXYGEN AND WITH ANOXIA
Amino acid nitrogen per 100 gm. of liver tissue -
Before After 60 After 120 After 180 After 240
immersion min min mn mn
mg mg mg mg mg
With oxygen 43.8 51.9 58.6 78.2 96.7
With anoxia 47.2 71.8 67.1 82.3 86.1
Liver slices have been immersed in Krebs-Ringer solution and gassed with oxygen and carbon dioxide as indicated
in Table 4 or with nitrogen and carbon dioxide as in Table 5. Averages for oxygenation were obtained from six
experiments and those for anoxia from four experiments.
Amino acid nitrogen in liver slices (Table 6) has been, in average, 43.4 per 100 gm.
with variation from 26.9 to 64.7 mg. Corresponding figures for urea (Table 4)
have been 33.7 mg per 100 gm. with variation from 14.9 to 50.1 mg. When the
medium is gassed with oxygen in the presence of carbon dioxide, amino acid nitro-
gen in tissue slices and fluid medium increases continuously at a time when urea is
increasing, but at a much slower rate, so that its quantity is slightly more than
doubled. During the first 60 min of immersion the amino acid nitrogen of the
Downloaded by guest on November 11, 2021482 BIOCHEMISTRY: E. L. OPIE PROC. N. A. S.
tissue slices diminishes rapidly and later maintains an even level. With anoxia
in the presence of nitrogen and carbon dioxide, amino acid nitrogen has increased
during four hr but the increase has been somewhat less than that with oxygenation.
Urea formation by liver tissue has been compared with that by kidney cortex
under the same conditions. In Table 7 are the average results of three experiments
TABLE 7
UREA FORMATION AND WATER MOVEMENT IN SLICES OF KIDNEY CORTEX WITH OXYGEN AND
wiTH ANOXIA
.-Urea-mg per 100 gm kidney cortex---. --Water-% of weight--.
Before After 120 After 240 Before After 120 After 240
immersion mn mm immersion min min
With oxygen 82.4 94.6 97.3 100 130.3 133.2
With nitrogen 82.4 96.4 105.3 100 138.3 139.7
Urea formation and water movement of kidney slices under conditions similar to those recorded for liver in
Table 6. Average figures are from three experiments in each of which corresponding slices have been from the
same animal.
in each of which slices of kidney cortex from the same animal have been exposed to
oxygen, 95 per cent with carbon dioxide 5 per cent, and to nitrogen with carbon
dioxide. The average quantity of urea in slices of kidney cortex has been much
greater than that in liver, i.e., an average of 33.7 mg per 100 gm. in liver (Table 4)
and 82.4 mg in kidney. The table shows that little, if any urea formation has oc-
curred. Intake of water by kidney cortex has been greater than that of liver under
similar conditions and, in contrast with liver, that with oxygenation and with anoxia
has been almost the same.
The experiments that have been described give evidence that liver slices immersed
in a suitable medium (Krebs-Ringer solution) at 380C, with inadequate oxygenation
or with anoxia, take up water in considerable quantity increasing during 3 or 4 hr.
On the contrary, with sufficient oxygenation aided by the presence of carbon dioxide,
water taken in within the first hr is later in part or wholly lost. The attempt has
been made in these and in earlier experiments to correlate the movement of water
with changes in intracellular osmotic pressure.
Special consideration has been given to the osmotic pressure maintained by liver
cells immediately after their removal from the body, for, as the result of this in-
tracellular pressure, liver cells are isotonic with a solution of sodium chloride with
molar concentration twice that of physiological salt solution or of blood plasma.
The increased level of isotonicity shown by autolysis of liver when tested in graded
solutions of sodium chloride has suggested that disintegration of protein may pro-
duce substances with smaller molecular weight capable of supplementing osmotic
pressure referable to electrolytes, chiefly those with potassium or sodium base.
Liver cells during a short supravital period act as efficient osmometers' and are
found to be isotonic with solutions of some nitrogeneous metabolites in molar con-
centration which approximates that of sodium chloride isotonic with these cells.
Tests by means of graded solutions have shown that liver cells are isotonic with
solutions of choline, arginine, and guanidine in concentrations approximating those
of sodium chloride isotonic with liver. It is perhaps significant that ornithine and
citrulline, associated with arginine in the production of urea (Table 3), are isotonic
with liver in concentrations approximating that of arginine. Urea, however, is
isotonic with liver only in highly concentrated solutions (approximately 1.5 molar)
and, being highly diffusible, readily leaves the cell.
Downloaded by guest on November 11, 2021VOL. 46, 1960 BIOCHEMISTRY: E. L. OPIE 483
The present experiments show that surviving liver cells with adequate oxygena-
tion produce urea from substances present in liver cells whereas with anoxia little
urea is formed. In the former instance, loss of water is associated with urea forma-
tion. When, on the contrary, urea formation is inhibited by anoxia, there is abun-
dant intake of water. It is noteworthy that the liver slices of these experiments con-
tain abundant amino acid nitrogen and this increases with incubation when oxy-
genated, and with anoxia.
Kidney cortex unlike liver tissue under corresponding conditions forms very little
urea with oxygenation or with anoxia, and water intake considerable with both, is
not inhibited by oxygenation.
Evidence has been presented to show that intracellular osmotic pressure is main-
tained not only by electrolytes with sodium or potassium base but by nitrogenous
products of metabolism as well. On the one hand, with inadequate oxygenation
nitrogenous substances which increase osmotic pressure accumulate in the cell and,
on the other, with adequate oxygenation they contribute to the formation of urea
which is readily diffusible and leaves the cell.
Accumulation of substances which maintain intracellular osmotic pressure has
been associated with the entrance of water into the liver cell. With formation of
urea and presumably with diminished osmotic pressure, water has left it. This zone
of osmotic activity, represented in vitro by adequate oxygenation at one extreme
and by anoxi. at the other, is, during life, favorable to the adjustment of functional
needs.
I Opie, E. L., "Isotonicity of liver and of kidney tissue in solutions of electrolytes," J. Exp.
Med., 110, 103 (1959).
2 Robinson, R. A., and R. H. Stokes, "Tables of osmotic and activity coefficients of electrolytes
in solution at 250C," Tr. Faraday Soc., 45, 612 (1949).
3Krebs, H. A., and K. Henseleit, "Untersuchen uber dies Harnstoffbildung im Tierkorper,"
Zeit. f. physiol. Chem., 210, 3 (1932).
4 Opie, E. L., and M. B. Rothbard, "Osmotic homeostasis maintained by mammalian liver,
kidney and other tissues," J. Exp. Med., 97, 483 (1953).
6 Opie, E. L., "Changes caused by injurious agents in the permeability of surviving cells of liver
and of kidney," J. Exp. Med., 104, 897 (1956).
6 Kossel, A., and H. D. Dakin, "Uber die arginase," Zeit. f. phpsiol. Chem., 41, 321 (1904); ibid.,
42, 181 (1904).
7Archibald, R. M., "Colorimetric determination of urea," J. Bil. Chem., 157, 507 (1945).
8 Folin, O., "A colorimetric determination of the amino acid nitrogen in normal blood," J. Biol.
Chem., 51, 377 (1922).
o Natelson, S., Microtechniques of Clinical Chemistry (Springfield, Ill.: Charles C Thomas,
1957).
Downloaded by guest on November 11, 2021You can also read
