- Email: [email protected]
Alteration of urine composition of the normal force-fed rat by an organic mercurial diuretic agent
TOXICOLOGY
AND
APPLIED
Alteration
3, 431-444
PHARMACOLOGY
of
Force-Fed
Urine Rat
Composition by
an
Diuretic JOHN
F.
VAN
PILSUM, LIDIA
Department
of Physiological
of the
Organic
Normal
Mercurial
Agent’ ELIZABETH
I(.
Chemistry, Minneapolis, Received
(1961)
Z.
WICKENS,
AND
Minnesota
Medical
FILONOWICH University Minnesota
December
of
School,
14, 1960
It is well known the urinary excretion of sodium and potassium from man and various experimental animals may be altered by the administration of certain organic mercurial compounds. Little information has been obtained about the composition of urine from animals that have been given these drugs. Such an investigation might indicate some mechanism for the drug-induced alteration of the sodium and potassium excretion and also might reveal some chemistry involved in the normal excretion of these two compounds. Force-fed rats were injected with an organic mercurial compound, N-( B-hydroxymercuri-y-methoxy)propyl-N’-succinylurea (Mercuhydrin), and 24-hour urine samples were collected daily for 10 days after the injection. Alterations were found in the daily urinary excretion of glucose, citric acid, organic acids, a-amino acid nitrogen, urea, ammonia, uric acid, creatine, arginine, undertermined nitrogen, total nitrogen, hydrogen ion, titratable acidity, sodium, potassium, and chloride. Total and inorganic sulfate, total and inorganic phosphate, total determined cations, and total determined anions remained essentially constant over the lo-day period. Kidneys from rats that had received the drug had normal transamidinase activity in vitro. The changes have been correlated and discussed. 1 These studies Minnesota: Grant Diseases, National States Department Science Foundation
were supported in part by the following grants to the University of No. A-2731 from the National Institute of Arthritis and Metabolic Institutes of Health, United States Public Health Service, United of Health, Education, and Welfare, and a grant from the National for research on guanidinium compound metabolism. 431
432
JOHN
F.
VAN
PILSUM
ET
AL.
METHODS
Nine adult, male, albino rats, weighing approximately 300 g were force fed to prevent contamination of the urine collections with scattered food and to ensure constant daily food intake. The diet contained: vitaminfree casein, 20 g; cornstarch, 55.53 g; corn oil, 20 g; choline chloride, 0.1 g; salt mixture no. 2,2 4 g; nL-methionine, 0.25 g; and Upjohn Zymadrops (a multiple vitamin preparation), 0.12 ml. The diet was blended with water in a Waring blendor, and the resultant slurry was made up to a total volume of 200 ml. The animals were started on the diet with a volume of 5 ml twice a day, increasing over a period of 5 days to 10 ml twice a day, with access to water ad libitum. It was found that the force-fed rats excreted creatine in their urine for about 2 weeks after being placed on this regimen and that the creatinuria ceased after this 2-week period. All animals, therefore, were force fed for a period of 2 weeks prior to any experimental procedures. The rats were divided into two groups (three controls and six experimental animals) ; each group was housed in a cage over a funnel and the urine was collected in bottles containing toluene as a preservative. Feces were prevented from entering the urine collection by a glass wool barrier in the mouth of the funnel. No attempts were made to measure the urine volume because of possible spillage of drinking water into the collecting bottles. The funnels were washed with distilled water and the control urine was made up to a volume of 250 ml, the experimental urine to a volume of 500 ml. The urines were analyzed for the following constituents according to published procedures: guanidine compounds (creatine, creatinine, arginine, and guanidinoacetic acid) (Van Pilsum et al., 1956), uric acid (Brown, 1945), glucose (Benedict, 191l), citric acid (Taussky and Shore, 1947), organic acids (Van Slyke and Palmer, 1920), urea nitrogen and ammonia nitrogen (Van Slyke and Cullen, 1914), total nitrogen (Lepper, 1950), a-amino acid nitrogen (Sobel et aE., 1957), total and inorganic sulfate (Folin, 1905-1906)) total and inorganic phosphate (Fiske and SubbaRow, 1925), chloride (Carr, 1951), titratable acidity (Henderson and Palmer, 1914), and mercury (Milton and Hoskins, 1947). Sodium and potassium were determined with a Coleman model no. 21 flame photometer, all pH measurementswere made with a Beckman pH meter, and all calorimetric measurementswith a Bausch and Lomb “Spec2 Salt Cleveland
mixture no. 28, Ohio.
2 was
purchased
from
Nutritional
Biochemical
Corporation,
EFFECT
OF MERCUHYDRIN
ON
URINE
COMPOSITION
433
tronic 20” calorimeter. A pH meter, instead of indicators, was used to determine end points in the determination of organic acids. Kidneys were measured for transamidinase activity by a previously reported procedure (Van Pilsum et al., 1957). Of Mercuhydrin, 0.02 ml (2.8 mg mecury per kilogram body weight) was found to be the minimum single dose necessary to produce an alteration in the urine sodium and potassium excretion and was administered intramuscularly to the six experimental animals; the urines were collected every 24 hours for 10 days following the injection. The urines from the control group were collected also every 24 hours for 10 days. The normal values in the tables were calculated as the average of the ten daily samples from the control group. These averages agreed well with measurements made on urines collected from the experimental group prior to injection of the drug. RESULTS
Eighty-five per cent of the mercury was recovered in the urine and was excreted in the first 4 days as 80, 2, 1, and 1% of the injected dose, respectively. The compounds excreted were expressedas millimoles or milliequivalents per kilogram body weight per 24 hours. The nitrogen-containing compounds were expressedas millimoles nitrogen and were tabulated as such (Table 1) and also as the percentage of the total nitrogen (Table 2). Any alteration from a normal value of more than -+ 15% was considered to be significant. The change from normal of the nitrogen compounds (Table 1) may be summarized as follows: Urea nitrogen was decreased the first 2 days (- 42% and - 16%). Ammonia nitrogen was decreased the first day (- 55%) and increased on the third day (+ 25%). a-Amino nitrogen was increased greatly the first 4 days (+ 240, + 250, + 170, and + 52%, respectively). Creatinine excretion was decreasedthe first day (- 40%)) and creatine appeared in the urine in large amounts the first 6 days. Arginine was increased greatly the first 3 days (f 390, + 390, and + 2100/o,respectively) and remained elevated for the lo-day period. Guanidinoacetic acid was increasedon days 2 to 6 (+ 28, + 28, + 57, + 57, and + 2870, respectively), and uric acid was decreasedthe first 4 days (- 40, - 70, - 60, and - 30%, respectively). The total nitrogen (Kjeldahl) decreasedthe first day (- 32%) and was increased on days 3 and 4 (+ 27 and + 2474). Undetermined nitrogen was increasedgreatly in the first 4 days (+ 640, + 440, + 1060, and + 12007,,
2
3
4
5
6
7
8
9 10
Day
Day
Day
Day
Day
Day
Day
Day Day
a Values:
28.1
Day
per
1.0
0.8
0.9
0.8
excreted
0.9
0.9
1.0
1.3
0.7
0.8
kilogram
1 .o
0.8
1.2
1.0
1.0
2.2
0.9
0.6
2.9
1 .o
2.7
0.06
0.2
0.2
0.3
0.4
0.5
0.20
0.06
body
< 0.06
< 0.06
< 0.06
<
<
Creatine
Creatinine
1
weight
0.06
0.06
0.07
per
24 hours.
0.07
0.07
0.07
0.08
0.09
0.08 0.08
0.11
0.11
0.07 0.09
0.09
0.14
0.09
0.22
0.20
0.20
0.18
0.18
0.18
0.18
0.14
0.08
0.06
0.20 0.12
Uric acid
MI.
o.ot
0.02
0.07
Guanidinoacetic
OF
0.22
0.05
Arginine
ADMINISTRATION
TABLE AFTER
COMPOSITION”
0.8
a-amino
NITROGEN
nitrogen
6.1
53.0
millimoles
6.8
5.6
5.6
6.1
6.1
7.4
8.3
6.0
3.0
6.7
NH+,
53.0
50.0
51.0
53.1
54.3
53.6
53.9
40.6
48.4
1
Normal
Urea
Day
URINE
61.1
61.7
62.3
0.6
< 0.5
57.5 57.5 62.4
2.4
< 0.5
61.1
58.7
0.6
0.7
6.6
62.3
63.5
5.8
2.6
3.8
< 0.5
Undetn.
61.7
62.8
70.1
71.9
66.1 63.5
53.9
51.3
38.7
56.5
57.2 34.9
Total detn.
Total (sum)
MERCUHYDRIN
2
3
4
5
6
7
8
9
10
Day
Day
Day
Day
Day
Day
Day
Day
Day
a Values:
72.6
Day
per cent total
85.9
85.0
87.0
83.4
85.2
85.5
76.4
75.0
75.4
85.6
1
Normal
Urea
Day
URINE
determined
9.9
10.9
9.8
9.2
9.8
9.6
10.5
11.5
11.1
7.8
11.8
NH+,
NITROGEN
nitrogen.
1.2
2.1
1.3
1.3
1.6
1.3
1.8
3.0
5.3
7.1
1.4
a-Amino
COMPOSITIONS
1.5
1.4
1.5
1.6
1.5
1.6
1.7
1.4
1.7
1.5
1.7
Creatinine
2
< 0.1
< 0.1
< 0.1
< 0.1
0.3
0.3
0.4
0.6
0.8
0.5
Creatine ---~ < 0.1
ADMINISTRATION
TABLE AFTER
0.02
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
0.6
0.6
0.08
Arginine
OF MI,
MERCUHYDRIN
0.1
0.1
0.1
0.1
0.2
0.2
0.2
0.1
0.2
0.2
0.1
Guanidinoacetic
0.3
0.3
0.3
0.3
0.3
0.3
0.2
0.1
0.1
0.3
0.4
Uric acid
0.9
co.9
< 0.9
3.9
1.0
1.1
9.2
8.1
5.0
9.5
<0.9
Undetn.
2 =; i: P
z
3 g
s
g
2 8 2
E a
% g
5
436
JOHN
F.
VAN
PILSUM
ET
AL.
respectively) and remained elevated through day 7. The per cent changes from normal calculated from the data in Table 1 are presentedin graphical form in Fig. 1. When the nitrogen compounds were expressed as percentages of total nitrogen (Table 2) the alteration of creatine, arginine, a-amino acid nitrogen, and undetermined nitrogen essentially were the same as when
CREATINE
UNDETERMINED ARGININE ALPHA AMINO CREATININE GUANIDINOACETIC URIC ACID AMMONIA UREA TOTAL
2 FIG. normal
1. The in the
4
6
data in Table 1 calculated excretion of the nitrogen
8
IO
and plotted compounds.
Days as the per
cent
change
from
expressedas the change from normal of each compound-that is, all were above normal the first 4-5 days. Uric acid excretion was below normal on days 2 to 6 (- 72, - 75, - 50, - 25, and - 25%, respectively). Urea excretion was below normal the first day (- 150/U) and ammonia was decreased34% the first day. Creatinine excretion was below normal on days 3 and 4 (- 17 and - 30% ) and guanidinoacetic acid was elevated approximately 100% on days 1, 2, 4, 5, and 6. The per cent changesfrom normal of the data in Table 2 are shown in Fig. 2.
EFFECT
OF
MERCUHYDRIN
ON
URINE
COMPOSITION
437
The excretion of glucose, citric acid, and titratable acidity is shown in Table 3. Glucose3 appeared in the urine the first four days and citric acid excretion was greatly increased the first 3 days (+ 450, + 200, and + loo%, respectively) and remained elevated for the IO-day period. Titratable acidity was elevated for the lo-day period (f 19, + 62, + 69, + 69, + 75, + 50, + 69, + 75, + 62, and + 755%,respectively).
CREATINE
UNDETERMINED ARGININE ALPHA AMINO CREATININE GUANIDINOACETIC URIC ACID AMMONIA UREA
FIG. 2. The data in Table 2 calculated as and plotted as the per cent change from normal of the excretion of the nitrogen compounds expressed as their per cent of the total determined nitrogen.
The data in Table 4 may be summarized as follows: Potassium excretion was increased the first 2 days (+ 160 and + 32%, respectively). Sodium excretion was increased 100% the first day and was decreasedon days 3 to 5 (- 28, - 47, and - 27% respectively). Hydrogen ion concentration was elevated for the IO-day period (+ 100, + 290, + 180, + 210, + 288, + 146, + 210, + 177, + 100, and + 146%). Chloride a The presence of glucose in the urine was confirmed by using gluccse which contain a specific glucose metabolizing enzyme, glucose oxidase.
test strips
438
JOHN
F.
VAN
PILSUM
ET
AL.
excretion was increased on the third and fourth days (+ 120 and + 45%), was decreased on days 5 to 8 (- 25, - 30, - 30, and - 355% ) and increased on days 9 and 10 (+ 40 and + 1870 ) . Organic acids were increased the first 3 days (+ 28, + 44, and + 2870, respectively) and decreased slightly the last 6 days. Total and inorganic sulfate and millimoles total and inorganic phosphate essentially were constant. The milliequivalents phosphate anions were calculated from the pH of each specimen. The total determined anions and cations essentially remained constant. The total determined anions was greater than the cations, and it is presumed that calcium plus magnesium would make up this deficit. TABLE URINE
COMPOSITION
AFTER
Normal Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Dav 10 millimoles
OF 0.02
excreted
<
0.5
< < < < < <
4.2 2.8 2.1 0.6 0.5 0.5 0.5 0.5 0.5 0.5
per
kilcgram
ML
Citric acid
Glucose
Day
a Values:
3
ADMINISTRATION
Titratable acidity
0.016 0.091 0.048 0.030 0.026 0.028 0.02 1 0.023 0.034 0.029 0.023 body
weight
MERCUHYDRIN
1.6 1.9 2.6 2.7 2.7 2.8 2.4 2.7 2.8 2.6 2.8 per
24 hours.
The ratio of cations to anions was 0.79, 0.70, 0.70, 0.67, 0.72, 0.77, 0.70, 0.74, 0.75, and 0.75 on days 1 through 10. The per cent changes from normal of the data in Tables 3 and 4 are shown in Fig. 3. The net effect over the IO-day period (Table 5) was a great increase in the urinary excretion of a-amino acid nitrogen, creatine, arginine, undetermined nitrogen, glucose, citric acid, true and titratable acidity, and potassium. The total lo-day excretion of all the other constituents was unchanged except uric acid which was lower than normal, and guanidinoacetic acid which was slightly above normal. It is known that kidney transamidinase is a --SH enzyme involved in the metabolism of arginine and creatine (Van Pilsum et al., 195 7)-two compounds that were excreted in large amounts as a result of the Mercuhydrin. A group of rats receiving fox chow and water ad libitum were
1.9 1.4
2 3
4 5
6 7
8 9 10
Day Day
Day Day
Day Day
Day Day Day
a Values:
1.4 3.7
Normal Day 1
2.0 1.9 1.8
1.8 1.9
1.0 1.3
1.9 1.3
1.8 3.7
Naf
milliequivalents
1.6 1.5 1.5
1.3 1.5
1.5 1.5
K+
Day
URINE
1.6 1.8
2.3 1.4
1.8 1.6
1.2 1.4
8.3 7.4
6.1 6.1
5.6 5.6
6.8 6.1
per
1.2 2.3
3.0 6.0
excreted
0.6
(X 10-d)
6.7
NH+,
Hf
ELECTROLYTE
kilogram
10.2 9.4
9.0 9.2
8.9 9.2
11.0 9.9
10.4 9.8
9.9
Total detn. cations
body
COMPOSITIONS
weight
1.5 1.3
0.8 0.7
0.8 0.8
2.4 1.6
1.0 1.0
1.1
Cl-
per
2.9 2.7
2.9 2.7
3.2 2.9
4.3 3.7
4.3 4.8
3.3
acids
24
Org.
Total
4.3 4.3
4.5 4.2
4.3 3.9
4.7 4.3
4.0 4.5
4.0
so,
hours.
TABLE 4 AFTER ADMINISTRATION ML
3.6
4.1
4.0
3.7 3.6 3.9 4.1 4.0
3.7 3.9 4.1 4.1
3.7 4.4 4.0 4.2
3.7
3.3 3.9 3.9
3.5 4.0 3.9
4.2 4.4 4.5
moles) 3.7 3.2
(mmoles) 3.9 3.3
4.0
Total
4.5
4.5 5.0 5.0 5.1
4.3
4.7 5 .o
3.7
4.1
anions 5.6
12.5
11.9 13.1 12.4 13.7
12.3
15.8 14.8
13.7
13.2
14.0
detn. anions
; z il z
8 K
2
s
g
;s z z
2 Lk
g
2 $
Inorg. PO,
8
Total PO,
Inorg. PO, Cm-
MERCUHYDRIN
3.8
so, 4.0
Inorg.
OF 0.02
E
r
440
JOHN
F.
VAN
PILSUM
ET
AL.
given 0.02 ml Mercuhydrin and were killed by a blow on the head at 0.083, 0.166, 0.25, 0.50, 1, 3, 6, 12, 24, 48, and 72 hours after the injection. Their kidneys were removed and analyzed for transamidinase activity in vitro. No change from normal was observed in kidneys from the rats that had received the Mercuhydrin.
CITRIC TITRAT-
I ( 3. The data change from normal FIG.
2
4
L I
6
8
, ,
IO
ACID ACIDITY
Doys
in Tables 3 and 4 calculated as and in the excretion of the compounds.
plotted
as the
per
cent
If the metabolism of creatine or arginine was not altered, at the kidney transamidinase step, perhaps the great increase in the excretion of the two compounds was the result of kidney tissue destruction by the mercurial. The creatine and arginine content of the kidney tissue homogenates (used for the transamidinase assay) were found to be unchanged. The abnormal rates of excretion of certain compounds may have been associatedwith altered serum levels. Rats receiving fox chow and water ad libitum were given a dose of 0.02 ml. Mercuhydrin, anesthetized with Nembutal, and killed by exsanguination 1, 2, 3, 4, 5, 6, 7, 8, 12, 15, 18,
EFFECT
OF
MERCUHYDRIN
ON
URINE
441
COMPOSITION
23, 26, 38, 40 and 44 hours after the injection. No change from normal levels was observed for creatine, creatinine, arginine, glucose, sodium, or potassium. It was established in a separate experiment that rats fed fox chow ad libitum responded to Mercuhydrin similarly to force-fed rats. TABLE ALTERATION
OF URINE
COMPOSITION
Compound Total nitrogen Urea nitrogen .4mmonia nitrogen
AFTER
5 ADMINISTRATION
Net lo-day
OF 0.02
change
mmoles/kg + 38.0 + 6.0 5.0
a-ilmino nitrogen Creatinine nitrogen Creatine nitrogen -4rginine nitrogen Guanidinoacetic nitrogen Uric acid nitrogen Undetermined nitrogen Glucose Citric acid
+ 6.0 0.6 + 1.4 + 0.6 + 0.14 0.5 + 18.0 + 7.6 + 0.20
Titratable acidity Potassium Sodium .4mmonium Hydrogen ion (X 10-4) Total determined cations Chloride Organic acids Total sulfate Inorganic sulfate Total phosphate Inorganic phosphate Inorganic phosphate Total determined anions
+ 10.0 + 3.0 + 0.4 5.0 + 10.7 1.8 + 1.0 + 1.0 + 2.6 + 1.4 O.ga 1.6a 9.1 6.1
ML
MERCUHYDRIN
% Change from normal + 6 + 1 7 + 74 6 + 230 + 130 + 20 25 +370 + 150 + 120
meqlkg + 60 + 21 + 2 7 + 200 2 + + + + -
9 3
6 4 2 4 17
4
a Millimoles
Salt mixture no. 2 contains two times as much potassium as sodium, and it was thought possible to prevent the loss of potassium in the urine as the result of the drug, by feeding a high-sodium, low-potassium diet. The salt mixture was modified by halving the potassium and doubling
442
JOHN
F.
VAN
PILSUM
ET
AL.
the sodium. Rats were force-fed the diet for 2 weeks before the injection of the mercurial. The sodium, potassium response as the result of this injection was similar to that observed in the rats force-fed the normal diet. There seemed to be a reciprocal relationship between potassium, sodium, and ammonium ions in rats given the mercurial. That is, when the potassium and sodium excretion increased, ammonium ion decreased and when sodium was retained, ammonium ion excretion increased. Rats were forcefed a diet containing 35% protein and 5’%, fat to furnish an excess of ammonia precursor. The high-protein rats demonstrated the same sodium and potassium response to the drug as the rats fed a normal diet. DISCUSSION
It is difficult to explain the drug-induced alteration of the urine composition on the basis of present day knowledge of kidney function. It seems reasonable, however, to discuss the effect of the drug as a metabolic inhibitor. The increased excretion of glucose, citric acid, organic acids, arginine, a-amino acid nitrogen, and undetermined nitrogen seems to indicate a decreased utilization of carbohydrate and protein at some place in the animal. The decrease in the excretion of urea and creatinine on day 1 could be the result of either a decreased glomerular filtration rate OY a block in the metabolism of protein and carbohydrate. For example, the great increase of the excretion of amino acids, undetermined nitrogen, and arginine, and the decrease of ammonia on day 1, may have reduced the amount of urea synthesized by the animal. Creatine may have appeared in the urine because it was not converted to creatine phosphate, and any decrease in the body stores of creatine phosphate should reduce the amount of creatinine formed and excreted. The below normal amounts of uric acid excreted also indicated a decrease in the metabolism of protein. An inhibition of the metabolism of protein also might explain the alteration in the excretion of the sodium, potassium, and chloride ions. Some correlations in the daily cation-anion excretion were as follows: The increased excretion of 4.2 meq of sodium plus potassium on the first day was accompanied by a decreased excretion of 3.7 meq of ammonium ion. The increased excretion of 1.3 meq of chloride on the third day was accompanied by an increased excretion of 1.6 meq of ammonium ion. The retention or decreased excretion of sodium on days 3 to 5 of 1.8 meq was accompanied by a net increased excretion of 1.7 meq of ammonium on those same 3 days. In other words, the daily excretion of total de-
EFFECT
OF
MERCUHYDRIN
ON
URINE
443
COMPOSITION
termined cations and total determined anions was remarkably constant. It is postulated, therefore, that the sodium and potassium may have been excreted in large amounts on day 1 because of a deficiency in the synthesis of ammonia. When the synthesis of ammonia had recovered, or even increased above normal, below normal amounts of sodium were excreted and also chloride was excreted in elevated amounts. SUMMARY Force-fed rats were injected with a single dose of an organic N-( 5-hydroxymercuri-y-methoxy) propyl-N’-succinylurea) , and samples were collected for 10 days following the injection.
mercurial compound, their 24-hour urine
The urinary excretion of inorganic and organic compounds was altered greatly. In spite of these alterations the total determined cations and total determined anions remained essentially constant as the result of the following: An increased excretion of sodium plus potassium on day 1 was accompanied by a decreased excretion of ammonium ion. An increase in the excretion of chloride on day 3 was accompanied by an increase in ammonium ion. A retention of sodium on days 3-5 was accompanied by an increased excretion of ammonium ion. The excretion of total and inorganic phosphate and total and inorganic sulfate remained essentially constant. There was an increase in the total IO-day excretion of creatine, glucose, citric acid, u-amino acid nitrogen, arginine, guanidinoacetic acid, undetermined nitrogen, titratable acidity, potassium, and hydrogen ion and a decrease of uric acid. The IO-day excretions of creatinine, urea, ammonia, sodium, total nitrogen, chloride, organic acids, sulfate, and phosphate were essentially unchanged. Kidney tissue from had normal amounts of creatine, creatinine, A mechanism chloride, sodium, been discussed.
rats that had been given a dose of the mercurial compound of creatine, arginine, and transamidinase activity. Serum levels arginine, glucose, sodium, and potassium also were not altered.
for the mercurial-induced potassium, and ammonium
alterations ions in
of the urinary excretion the normal force-fed rat
of has
REFERENCES BENEDICT,
Med.
(1911). The detection 57, 1193-1194. (1945). The determination
S. R.
Assoc.
and
estimation
of glucose
in urine.
J. Am.
H. of uric acid in human blood. J. Biol. Chem. 158, 601-608. CARR, C. W. (1951). The amperometric titration of plasma chloride and urine chloride. Arch. Biochem. Biophys. 34, 299-304. FISKE, C. H., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. FOLIN, 0. (1905-1906). On sulphate and sulphur determinations. J. Biol. Chem. 1, BROWN,
131-159.
HENDERSOS, excretion.
I. /.
J., and PALMER, W. W. Biol. Chem. 17, 305-315.
(1914).
On
the
several
factors
of acid
444
JOHN
LEPPER, H. A., ed. (1950). Agyicultuyal Chemists,
7th
Analyst
urine.
mination VAN PILSUM, of
creatine,
H.
A., of
R. J., nitrogen and
citric J. F.,
CHIAMORI, in urine.
SHORE,
E.
ET
AL.
of Analysis
of the Association
of Oficial
Association of Official Agricultural Station, Washington 4, D.C. The estimation of traces of mercury
of kidney D. D., and
of organic
N.,
and
Biol. D.
determination and PALMER, in urine.
(1957).
Med.
Determination
95, 808-813.
method
169, 103-118. E., and HESS, acid,
J.
for
(1956)
guanidine,
the
deter-
Determination and
methylguani-
222, 225-236.
and
WOLIN,
Pyoc.
Sot.
(1914).
of urea. W. W. /.
Biol.
A microcolorimetric
Chem. A.,
M.
Erptl.
guanidinoacetic
transamidinase. CULLEN, G. E.
acids
SEGALOVE,
PYOC. Sot.
(1947).
arginine,
fluids. /. F., BERMAN,
and its use in the VAN SLYKE, D. D., titration
Methods
acid. J. Biol. Chem. MARTIN, R. P., KITO,
creatinine,
dine in biological VAN PILSUM, J. properties VAN SLYKE,
Oficial
PILSUM
72, 6-10.
SOBEL, C., HENRY, of a-amino acid TAUSSKY,
VAN
ed., pp. 745-747. P. 0. Box 540, Benjamin Franklin F., and HOSIZINS, J. L. (1947).
Chemists, MILTON, R. in
F.
Biol.
E.
A.
Exptl.
(1957).
Med.
19,
211-228.
A permanent
J. Biol. (1920).
Chem.
Chem.
Assay
Biol.
preparation
Studies of 41, 567-585.
acidosis.
and
some
95, 96.100. of XVI.
urease, The
AND
APPLIED
Alteration
3, 431-444
PHARMACOLOGY
of
Force-Fed
Urine Rat
Composition by
an
Diuretic JOHN
F.
VAN
PILSUM, LIDIA
Department
of Physiological
of the
Organic
Normal
Mercurial
Agent’ ELIZABETH
I(.
Chemistry, Minneapolis, Received
(1961)
Z.
WICKENS,
AND
Minnesota
Medical
FILONOWICH University Minnesota
December
of
School,
14, 1960
It is well known the urinary excretion of sodium and potassium from man and various experimental animals may be altered by the administration of certain organic mercurial compounds. Little information has been obtained about the composition of urine from animals that have been given these drugs. Such an investigation might indicate some mechanism for the drug-induced alteration of the sodium and potassium excretion and also might reveal some chemistry involved in the normal excretion of these two compounds. Force-fed rats were injected with an organic mercurial compound, N-( B-hydroxymercuri-y-methoxy)propyl-N’-succinylurea (Mercuhydrin), and 24-hour urine samples were collected daily for 10 days after the injection. Alterations were found in the daily urinary excretion of glucose, citric acid, organic acids, a-amino acid nitrogen, urea, ammonia, uric acid, creatine, arginine, undertermined nitrogen, total nitrogen, hydrogen ion, titratable acidity, sodium, potassium, and chloride. Total and inorganic sulfate, total and inorganic phosphate, total determined cations, and total determined anions remained essentially constant over the lo-day period. Kidneys from rats that had received the drug had normal transamidinase activity in vitro. The changes have been correlated and discussed. 1 These studies Minnesota: Grant Diseases, National States Department Science Foundation
were supported in part by the following grants to the University of No. A-2731 from the National Institute of Arthritis and Metabolic Institutes of Health, United States Public Health Service, United of Health, Education, and Welfare, and a grant from the National for research on guanidinium compound metabolism. 431
432
JOHN
F.
VAN
PILSUM
ET
AL.
METHODS
Nine adult, male, albino rats, weighing approximately 300 g were force fed to prevent contamination of the urine collections with scattered food and to ensure constant daily food intake. The diet contained: vitaminfree casein, 20 g; cornstarch, 55.53 g; corn oil, 20 g; choline chloride, 0.1 g; salt mixture no. 2,2 4 g; nL-methionine, 0.25 g; and Upjohn Zymadrops (a multiple vitamin preparation), 0.12 ml. The diet was blended with water in a Waring blendor, and the resultant slurry was made up to a total volume of 200 ml. The animals were started on the diet with a volume of 5 ml twice a day, increasing over a period of 5 days to 10 ml twice a day, with access to water ad libitum. It was found that the force-fed rats excreted creatine in their urine for about 2 weeks after being placed on this regimen and that the creatinuria ceased after this 2-week period. All animals, therefore, were force fed for a period of 2 weeks prior to any experimental procedures. The rats were divided into two groups (three controls and six experimental animals) ; each group was housed in a cage over a funnel and the urine was collected in bottles containing toluene as a preservative. Feces were prevented from entering the urine collection by a glass wool barrier in the mouth of the funnel. No attempts were made to measure the urine volume because of possible spillage of drinking water into the collecting bottles. The funnels were washed with distilled water and the control urine was made up to a volume of 250 ml, the experimental urine to a volume of 500 ml. The urines were analyzed for the following constituents according to published procedures: guanidine compounds (creatine, creatinine, arginine, and guanidinoacetic acid) (Van Pilsum et al., 1956), uric acid (Brown, 1945), glucose (Benedict, 191l), citric acid (Taussky and Shore, 1947), organic acids (Van Slyke and Palmer, 1920), urea nitrogen and ammonia nitrogen (Van Slyke and Cullen, 1914), total nitrogen (Lepper, 1950), a-amino acid nitrogen (Sobel et aE., 1957), total and inorganic sulfate (Folin, 1905-1906)) total and inorganic phosphate (Fiske and SubbaRow, 1925), chloride (Carr, 1951), titratable acidity (Henderson and Palmer, 1914), and mercury (Milton and Hoskins, 1947). Sodium and potassium were determined with a Coleman model no. 21 flame photometer, all pH measurementswere made with a Beckman pH meter, and all calorimetric measurementswith a Bausch and Lomb “Spec2 Salt Cleveland
mixture no. 28, Ohio.
2 was
purchased
from
Nutritional
Biochemical
Corporation,
EFFECT
OF MERCUHYDRIN
ON
URINE
COMPOSITION
433
tronic 20” calorimeter. A pH meter, instead of indicators, was used to determine end points in the determination of organic acids. Kidneys were measured for transamidinase activity by a previously reported procedure (Van Pilsum et al., 1957). Of Mercuhydrin, 0.02 ml (2.8 mg mecury per kilogram body weight) was found to be the minimum single dose necessary to produce an alteration in the urine sodium and potassium excretion and was administered intramuscularly to the six experimental animals; the urines were collected every 24 hours for 10 days following the injection. The urines from the control group were collected also every 24 hours for 10 days. The normal values in the tables were calculated as the average of the ten daily samples from the control group. These averages agreed well with measurements made on urines collected from the experimental group prior to injection of the drug. RESULTS
Eighty-five per cent of the mercury was recovered in the urine and was excreted in the first 4 days as 80, 2, 1, and 1% of the injected dose, respectively. The compounds excreted were expressedas millimoles or milliequivalents per kilogram body weight per 24 hours. The nitrogen-containing compounds were expressedas millimoles nitrogen and were tabulated as such (Table 1) and also as the percentage of the total nitrogen (Table 2). Any alteration from a normal value of more than -+ 15% was considered to be significant. The change from normal of the nitrogen compounds (Table 1) may be summarized as follows: Urea nitrogen was decreased the first 2 days (- 42% and - 16%). Ammonia nitrogen was decreased the first day (- 55%) and increased on the third day (+ 25%). a-Amino nitrogen was increased greatly the first 4 days (+ 240, + 250, + 170, and + 52%, respectively). Creatinine excretion was decreasedthe first day (- 40%)) and creatine appeared in the urine in large amounts the first 6 days. Arginine was increased greatly the first 3 days (f 390, + 390, and + 2100/o,respectively) and remained elevated for the lo-day period. Guanidinoacetic acid was increasedon days 2 to 6 (+ 28, + 28, + 57, + 57, and + 2870, respectively), and uric acid was decreasedthe first 4 days (- 40, - 70, - 60, and - 30%, respectively). The total nitrogen (Kjeldahl) decreasedthe first day (- 32%) and was increased on days 3 and 4 (+ 27 and + 2474). Undetermined nitrogen was increasedgreatly in the first 4 days (+ 640, + 440, + 1060, and + 12007,,
2
3
4
5
6
7
8
9 10
Day
Day
Day
Day
Day
Day
Day
Day Day
a Values:
28.1
Day
per
1.0
0.8
0.9
0.8
excreted
0.9
0.9
1.0
1.3
0.7
0.8
kilogram
1 .o
0.8
1.2
1.0
1.0
2.2
0.9
0.6
2.9
1 .o
2.7
0.06
0.2
0.2
0.3
0.4
0.5
0.20
0.06
body
< 0.06
< 0.06
< 0.06
<
<
Creatine
Creatinine
1
weight
0.06
0.06
0.07
per
24 hours.
0.07
0.07
0.07
0.08
0.09
0.08 0.08
0.11
0.11
0.07 0.09
0.09
0.14
0.09
0.22
0.20
0.20
0.18
0.18
0.18
0.18
0.14
0.08
0.06
0.20 0.12
Uric acid
MI.
o.ot
0.02
0.07
Guanidinoacetic
OF
0.22
0.05
Arginine
ADMINISTRATION
TABLE AFTER
COMPOSITION”
0.8
a-amino
NITROGEN
nitrogen
6.1
53.0
millimoles
6.8
5.6
5.6
6.1
6.1
7.4
8.3
6.0
3.0
6.7
NH+,
53.0
50.0
51.0
53.1
54.3
53.6
53.9
40.6
48.4
1
Normal
Urea
Day
URINE
61.1
61.7
62.3
0.6
< 0.5
57.5 57.5 62.4
2.4
< 0.5
61.1
58.7
0.6
0.7
6.6
62.3
63.5
5.8
2.6
3.8
< 0.5
Undetn.
61.7
62.8
70.1
71.9
66.1 63.5
53.9
51.3
38.7
56.5
57.2 34.9
Total detn.
Total (sum)
MERCUHYDRIN
2
3
4
5
6
7
8
9
10
Day
Day
Day
Day
Day
Day
Day
Day
Day
a Values:
72.6
Day
per cent total
85.9
85.0
87.0
83.4
85.2
85.5
76.4
75.0
75.4
85.6
1
Normal
Urea
Day
URINE
determined
9.9
10.9
9.8
9.2
9.8
9.6
10.5
11.5
11.1
7.8
11.8
NH+,
NITROGEN
nitrogen.
1.2
2.1
1.3
1.3
1.6
1.3
1.8
3.0
5.3
7.1
1.4
a-Amino
COMPOSITIONS
1.5
1.4
1.5
1.6
1.5
1.6
1.7
1.4
1.7
1.5
1.7
Creatinine
2
< 0.1
< 0.1
< 0.1
< 0.1
0.3
0.3
0.4
0.6
0.8
0.5
Creatine ---~ < 0.1
ADMINISTRATION
TABLE AFTER
0.02
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
0.6
0.6
0.08
Arginine
OF MI,
MERCUHYDRIN
0.1
0.1
0.1
0.1
0.2
0.2
0.2
0.1
0.2
0.2
0.1
Guanidinoacetic
0.3
0.3
0.3
0.3
0.3
0.3
0.2
0.1
0.1
0.3
0.4
Uric acid
0.9
co.9
< 0.9
3.9
1.0
1.1
9.2
8.1
5.0
9.5
<0.9
Undetn.
2 =; i: P
z
3 g
s
g
2 8 2
E a
% g
5
436
JOHN
F.
VAN
PILSUM
ET
AL.
respectively) and remained elevated through day 7. The per cent changes from normal calculated from the data in Table 1 are presentedin graphical form in Fig. 1. When the nitrogen compounds were expressed as percentages of total nitrogen (Table 2) the alteration of creatine, arginine, a-amino acid nitrogen, and undetermined nitrogen essentially were the same as when
CREATINE
UNDETERMINED ARGININE ALPHA AMINO CREATININE GUANIDINOACETIC URIC ACID AMMONIA UREA TOTAL
2 FIG. normal
1. The in the
4
6
data in Table 1 calculated excretion of the nitrogen
8
IO
and plotted compounds.
Days as the per
cent
change
from
expressedas the change from normal of each compound-that is, all were above normal the first 4-5 days. Uric acid excretion was below normal on days 2 to 6 (- 72, - 75, - 50, - 25, and - 25%, respectively). Urea excretion was below normal the first day (- 150/U) and ammonia was decreased34% the first day. Creatinine excretion was below normal on days 3 and 4 (- 17 and - 30% ) and guanidinoacetic acid was elevated approximately 100% on days 1, 2, 4, 5, and 6. The per cent changesfrom normal of the data in Table 2 are shown in Fig. 2.
EFFECT
OF
MERCUHYDRIN
ON
URINE
COMPOSITION
437
The excretion of glucose, citric acid, and titratable acidity is shown in Table 3. Glucose3 appeared in the urine the first four days and citric acid excretion was greatly increased the first 3 days (+ 450, + 200, and + loo%, respectively) and remained elevated for the IO-day period. Titratable acidity was elevated for the lo-day period (f 19, + 62, + 69, + 69, + 75, + 50, + 69, + 75, + 62, and + 755%,respectively).
CREATINE
UNDETERMINED ARGININE ALPHA AMINO CREATININE GUANIDINOACETIC URIC ACID AMMONIA UREA
FIG. 2. The data in Table 2 calculated as and plotted as the per cent change from normal of the excretion of the nitrogen compounds expressed as their per cent of the total determined nitrogen.
The data in Table 4 may be summarized as follows: Potassium excretion was increased the first 2 days (+ 160 and + 32%, respectively). Sodium excretion was increased 100% the first day and was decreasedon days 3 to 5 (- 28, - 47, and - 27% respectively). Hydrogen ion concentration was elevated for the IO-day period (+ 100, + 290, + 180, + 210, + 288, + 146, + 210, + 177, + 100, and + 146%). Chloride a The presence of glucose in the urine was confirmed by using gluccse which contain a specific glucose metabolizing enzyme, glucose oxidase.
test strips
438
JOHN
F.
VAN
PILSUM
ET
AL.
excretion was increased on the third and fourth days (+ 120 and + 45%), was decreased on days 5 to 8 (- 25, - 30, - 30, and - 355% ) and increased on days 9 and 10 (+ 40 and + 1870 ) . Organic acids were increased the first 3 days (+ 28, + 44, and + 2870, respectively) and decreased slightly the last 6 days. Total and inorganic sulfate and millimoles total and inorganic phosphate essentially were constant. The milliequivalents phosphate anions were calculated from the pH of each specimen. The total determined anions and cations essentially remained constant. The total determined anions was greater than the cations, and it is presumed that calcium plus magnesium would make up this deficit. TABLE URINE
COMPOSITION
AFTER
Normal Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Dav 10 millimoles
OF 0.02
excreted
<
0.5
< < < < < <
4.2 2.8 2.1 0.6 0.5 0.5 0.5 0.5 0.5 0.5
per
kilcgram
ML
Citric acid
Glucose
Day
a Values:
3
ADMINISTRATION
Titratable acidity
0.016 0.091 0.048 0.030 0.026 0.028 0.02 1 0.023 0.034 0.029 0.023 body
weight
MERCUHYDRIN
1.6 1.9 2.6 2.7 2.7 2.8 2.4 2.7 2.8 2.6 2.8 per
24 hours.
The ratio of cations to anions was 0.79, 0.70, 0.70, 0.67, 0.72, 0.77, 0.70, 0.74, 0.75, and 0.75 on days 1 through 10. The per cent changes from normal of the data in Tables 3 and 4 are shown in Fig. 3. The net effect over the IO-day period (Table 5) was a great increase in the urinary excretion of a-amino acid nitrogen, creatine, arginine, undetermined nitrogen, glucose, citric acid, true and titratable acidity, and potassium. The total lo-day excretion of all the other constituents was unchanged except uric acid which was lower than normal, and guanidinoacetic acid which was slightly above normal. It is known that kidney transamidinase is a --SH enzyme involved in the metabolism of arginine and creatine (Van Pilsum et al., 195 7)-two compounds that were excreted in large amounts as a result of the Mercuhydrin. A group of rats receiving fox chow and water ad libitum were
1.9 1.4
2 3
4 5
6 7
8 9 10
Day Day
Day Day
Day Day
Day Day Day
a Values:
1.4 3.7
Normal Day 1
2.0 1.9 1.8
1.8 1.9
1.0 1.3
1.9 1.3
1.8 3.7
Naf
milliequivalents
1.6 1.5 1.5
1.3 1.5
1.5 1.5
K+
Day
URINE
1.6 1.8
2.3 1.4
1.8 1.6
1.2 1.4
8.3 7.4
6.1 6.1
5.6 5.6
6.8 6.1
per
1.2 2.3
3.0 6.0
excreted
0.6
(X 10-d)
6.7
NH+,
Hf
ELECTROLYTE
kilogram
10.2 9.4
9.0 9.2
8.9 9.2
11.0 9.9
10.4 9.8
9.9
Total detn. cations
body
COMPOSITIONS
weight
1.5 1.3
0.8 0.7
0.8 0.8
2.4 1.6
1.0 1.0
1.1
Cl-
per
2.9 2.7
2.9 2.7
3.2 2.9
4.3 3.7
4.3 4.8
3.3
acids
24
Org.
Total
4.3 4.3
4.5 4.2
4.3 3.9
4.7 4.3
4.0 4.5
4.0
so,
hours.
TABLE 4 AFTER ADMINISTRATION ML
3.6
4.1
4.0
3.7 3.6 3.9 4.1 4.0
3.7 3.9 4.1 4.1
3.7 4.4 4.0 4.2
3.7
3.3 3.9 3.9
3.5 4.0 3.9
4.2 4.4 4.5
moles) 3.7 3.2
(mmoles) 3.9 3.3
4.0
Total
4.5
4.5 5.0 5.0 5.1
4.3
4.7 5 .o
3.7
4.1
anions 5.6
12.5
11.9 13.1 12.4 13.7
12.3
15.8 14.8
13.7
13.2
14.0
detn. anions
; z il z
8 K
2
s
g
;s z z
2 Lk
g
2 $
Inorg. PO,
8
Total PO,
Inorg. PO, Cm-
MERCUHYDRIN
3.8
so, 4.0
Inorg.
OF 0.02
E
r
440
JOHN
F.
VAN
PILSUM
ET
AL.
given 0.02 ml Mercuhydrin and were killed by a blow on the head at 0.083, 0.166, 0.25, 0.50, 1, 3, 6, 12, 24, 48, and 72 hours after the injection. Their kidneys were removed and analyzed for transamidinase activity in vitro. No change from normal was observed in kidneys from the rats that had received the Mercuhydrin.
CITRIC TITRAT-
I ( 3. The data change from normal FIG.
2
4
L I
6
8
, ,
IO
ACID ACIDITY
Doys
in Tables 3 and 4 calculated as and in the excretion of the compounds.
plotted
as the
per
cent
If the metabolism of creatine or arginine was not altered, at the kidney transamidinase step, perhaps the great increase in the excretion of the two compounds was the result of kidney tissue destruction by the mercurial. The creatine and arginine content of the kidney tissue homogenates (used for the transamidinase assay) were found to be unchanged. The abnormal rates of excretion of certain compounds may have been associatedwith altered serum levels. Rats receiving fox chow and water ad libitum were given a dose of 0.02 ml. Mercuhydrin, anesthetized with Nembutal, and killed by exsanguination 1, 2, 3, 4, 5, 6, 7, 8, 12, 15, 18,
EFFECT
OF
MERCUHYDRIN
ON
URINE
441
COMPOSITION
23, 26, 38, 40 and 44 hours after the injection. No change from normal levels was observed for creatine, creatinine, arginine, glucose, sodium, or potassium. It was established in a separate experiment that rats fed fox chow ad libitum responded to Mercuhydrin similarly to force-fed rats. TABLE ALTERATION
OF URINE
COMPOSITION
Compound Total nitrogen Urea nitrogen .4mmonia nitrogen
AFTER
5 ADMINISTRATION
Net lo-day
OF 0.02
change
mmoles/kg + 38.0 + 6.0 5.0
a-ilmino nitrogen Creatinine nitrogen Creatine nitrogen -4rginine nitrogen Guanidinoacetic nitrogen Uric acid nitrogen Undetermined nitrogen Glucose Citric acid
+ 6.0 0.6 + 1.4 + 0.6 + 0.14 0.5 + 18.0 + 7.6 + 0.20
Titratable acidity Potassium Sodium .4mmonium Hydrogen ion (X 10-4) Total determined cations Chloride Organic acids Total sulfate Inorganic sulfate Total phosphate Inorganic phosphate Inorganic phosphate Total determined anions
+ 10.0 + 3.0 + 0.4 5.0 + 10.7 1.8 + 1.0 + 1.0 + 2.6 + 1.4 O.ga 1.6a 9.1 6.1
ML
MERCUHYDRIN
% Change from normal + 6 + 1 7 + 74 6 + 230 + 130 + 20 25 +370 + 150 + 120
meqlkg + 60 + 21 + 2 7 + 200 2 + + + + -
9 3
6 4 2 4 17
4
a Millimoles
Salt mixture no. 2 contains two times as much potassium as sodium, and it was thought possible to prevent the loss of potassium in the urine as the result of the drug, by feeding a high-sodium, low-potassium diet. The salt mixture was modified by halving the potassium and doubling
442
JOHN
F.
VAN
PILSUM
ET
AL.
the sodium. Rats were force-fed the diet for 2 weeks before the injection of the mercurial. The sodium, potassium response as the result of this injection was similar to that observed in the rats force-fed the normal diet. There seemed to be a reciprocal relationship between potassium, sodium, and ammonium ions in rats given the mercurial. That is, when the potassium and sodium excretion increased, ammonium ion decreased and when sodium was retained, ammonium ion excretion increased. Rats were forcefed a diet containing 35% protein and 5’%, fat to furnish an excess of ammonia precursor. The high-protein rats demonstrated the same sodium and potassium response to the drug as the rats fed a normal diet. DISCUSSION
It is difficult to explain the drug-induced alteration of the urine composition on the basis of present day knowledge of kidney function. It seems reasonable, however, to discuss the effect of the drug as a metabolic inhibitor. The increased excretion of glucose, citric acid, organic acids, arginine, a-amino acid nitrogen, and undetermined nitrogen seems to indicate a decreased utilization of carbohydrate and protein at some place in the animal. The decrease in the excretion of urea and creatinine on day 1 could be the result of either a decreased glomerular filtration rate OY a block in the metabolism of protein and carbohydrate. For example, the great increase of the excretion of amino acids, undetermined nitrogen, and arginine, and the decrease of ammonia on day 1, may have reduced the amount of urea synthesized by the animal. Creatine may have appeared in the urine because it was not converted to creatine phosphate, and any decrease in the body stores of creatine phosphate should reduce the amount of creatinine formed and excreted. The below normal amounts of uric acid excreted also indicated a decrease in the metabolism of protein. An inhibition of the metabolism of protein also might explain the alteration in the excretion of the sodium, potassium, and chloride ions. Some correlations in the daily cation-anion excretion were as follows: The increased excretion of 4.2 meq of sodium plus potassium on the first day was accompanied by a decreased excretion of 3.7 meq of ammonium ion. The increased excretion of 1.3 meq of chloride on the third day was accompanied by an increased excretion of 1.6 meq of ammonium ion. The retention or decreased excretion of sodium on days 3 to 5 of 1.8 meq was accompanied by a net increased excretion of 1.7 meq of ammonium on those same 3 days. In other words, the daily excretion of total de-
EFFECT
OF
MERCUHYDRIN
ON
URINE
443
COMPOSITION
termined cations and total determined anions was remarkably constant. It is postulated, therefore, that the sodium and potassium may have been excreted in large amounts on day 1 because of a deficiency in the synthesis of ammonia. When the synthesis of ammonia had recovered, or even increased above normal, below normal amounts of sodium were excreted and also chloride was excreted in elevated amounts. SUMMARY Force-fed rats were injected with a single dose of an organic N-( 5-hydroxymercuri-y-methoxy) propyl-N’-succinylurea) , and samples were collected for 10 days following the injection.
mercurial compound, their 24-hour urine
The urinary excretion of inorganic and organic compounds was altered greatly. In spite of these alterations the total determined cations and total determined anions remained essentially constant as the result of the following: An increased excretion of sodium plus potassium on day 1 was accompanied by a decreased excretion of ammonium ion. An increase in the excretion of chloride on day 3 was accompanied by an increase in ammonium ion. A retention of sodium on days 3-5 was accompanied by an increased excretion of ammonium ion. The excretion of total and inorganic phosphate and total and inorganic sulfate remained essentially constant. There was an increase in the total IO-day excretion of creatine, glucose, citric acid, u-amino acid nitrogen, arginine, guanidinoacetic acid, undetermined nitrogen, titratable acidity, potassium, and hydrogen ion and a decrease of uric acid. The IO-day excretions of creatinine, urea, ammonia, sodium, total nitrogen, chloride, organic acids, sulfate, and phosphate were essentially unchanged. Kidney tissue from had normal amounts of creatine, creatinine, A mechanism chloride, sodium, been discussed.
rats that had been given a dose of the mercurial compound of creatine, arginine, and transamidinase activity. Serum levels arginine, glucose, sodium, and potassium also were not altered.
for the mercurial-induced potassium, and ammonium
alterations ions in
of the urinary excretion the normal force-fed rat
of has
REFERENCES BENEDICT,
Med.
(1911). The detection 57, 1193-1194. (1945). The determination
S. R.
Assoc.
and
estimation
of glucose
in urine.
J. Am.
H. of uric acid in human blood. J. Biol. Chem. 158, 601-608. CARR, C. W. (1951). The amperometric titration of plasma chloride and urine chloride. Arch. Biochem. Biophys. 34, 299-304. FISKE, C. H., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. FOLIN, 0. (1905-1906). On sulphate and sulphur determinations. J. Biol. Chem. 1, BROWN,
131-159.
HENDERSOS, excretion.
I. /.
J., and PALMER, W. W. Biol. Chem. 17, 305-315.
(1914).
On
the
several
factors
of acid
444
JOHN
LEPPER, H. A., ed. (1950). Agyicultuyal Chemists,
7th
Analyst
urine.
mination VAN PILSUM, of
creatine,
H.
A., of
R. J., nitrogen and
citric J. F.,
CHIAMORI, in urine.
SHORE,
E.
ET
AL.
of Analysis
of the Association
of Oficial
Association of Official Agricultural Station, Washington 4, D.C. The estimation of traces of mercury
of kidney D. D., and
of organic
N.,
and
Biol. D.
determination and PALMER, in urine.
(1957).
Med.
Determination
95, 808-813.
method
169, 103-118. E., and HESS, acid,
J.
for
(1956)
guanidine,
the
deter-
Determination and
methylguani-
222, 225-236.
and
WOLIN,
Pyoc.
Sot.
(1914).
of urea. W. W. /.
Biol.
A microcolorimetric
Chem. A.,
M.
Erptl.
guanidinoacetic
transamidinase. CULLEN, G. E.
acids
SEGALOVE,
PYOC. Sot.
(1947).
arginine,
fluids. /. F., BERMAN,
and its use in the VAN SLYKE, D. D., titration
Methods
acid. J. Biol. Chem. MARTIN, R. P., KITO,
creatinine,
dine in biological VAN PILSUM, J. properties VAN SLYKE,
Oficial
PILSUM
72, 6-10.
SOBEL, C., HENRY, of a-amino acid TAUSSKY,
VAN
ed., pp. 745-747. P. 0. Box 540, Benjamin Franklin F., and HOSIZINS, J. L. (1947).
Chemists, MILTON, R. in
F.
Biol.
E.
A.
Exptl.
(1957).
Med.
19,
211-228.
A permanent
J. Biol. (1920).
Chem.
Chem.
Assay
Biol.
preparation
Studies of 41, 567-585.
acidosis.
and
some
95, 96.100. of XVI.
urease, The