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Urea formation in the pregnant rat
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, l-9 (1957)
Urea Formation in the Pregnant Rat George H. Beaton From the Department of P-ublic Health Nutrition, University of Toronto, Toronto, Ontario
Received August 2, 1956
INTRODUCTION
A decreasein fasting blood urea is characteristic of human pregnancy While Nice (2) has stated that this is due to an increase in urea excretion, other workers (3) have found evidence of no change in urea clearance during pregnancy. In pregnant rats, the fasting blood urea either remains unchanged or is slightly increased. A decreased rate of urea production by slices of liver from pregnant rats has been found in uitro (4). It would be expected that the fetal demand for amino acids might be reflected by changes in amino acid catabolism in the maternal body. There could be a decreased formation of urea consequent to a lessened supply of amino acids for catabolism. This could explain the decreasedfasting blood urea of human pregnancy, but would not appear to be consistent with the unchanged blood urea in the pregnant rat. As part of a study of protein metabolism in pregnancy, it was decided to investigate urea formation, urea excretion, and the activities of several enzymes involved in urea formation. It has been possible to demonstrate that pregnant rats show a decreased formation and impaired excretion of urea. (1).
METHODS In all of the experiments to be described, female Wistar strain rats from t.he Carworth Farms colony were used. For breeding, the females were housed with males of the same strain, and vaginal smears were examined each morning; the day on which sperm was detected in the smear was taken as being day 0 of gestation. Following breeding, the pregnant and comparable nonpregnant control rats were housed in individual screen-bottomed cages in a room maintained at 75 f 2°F. and provided ad libitum with a 20yo corn oil, 20% casein diet. The diet con1
2
GEORGE
H.
BEATON
tained the following constituents in per cent by weight: casein 16, vitamin powder 4, corn oil 20, sucrose 54, nonnutritive cellulose (Alphacel) 2, salt mixture 4 [Steenbock-Nelson salts 140 (5)], choline 0.2, inositol 0.2, cod liver oil concentrate,’ 0.015. The vitamin powder was prepared by mixing thoroughly 800 g. casein with the following amounts (in milligrams) of vitamins: thiamine hydrochloride 100, riboflavine hydrochloride 100, pyridoxine hydrochloride 100, calcium pantothenate 400, nicotinic acid 400, p-aminobenzoic acid 400, biotin 20, folic acid 20. In the last experiment, vitamin E in the form of wheat germ oil* was added to the diet at a level of 10 mg./lOOO g. diet. The first three studies involved the intraperitoneal injection of various metabolites in pregnant and nonpregnant rats. In each case, the rats were fasted for 16-18 hr. prior to treatment. The initial control groups were injected with comparable volumes of 0.9% saline and killed immediately. The other groups were killed at the intervals indicated in the tables, following the injection of the metabolite. In the first experiment, on-alanine was administered as a 7.6% aqueous solution in an amount to provide 0.24 mg. nitrogen/g. body weight. Casein hydrolyaate’ was injected as an aqueous solution at a level of 0.36 mg. total nitrogen and 0.23 mg. amino nitrogen/g. of body weight. Both of these solutions were neutralized to pH 7.0 before injection. The third study involved the administration of a 4% solution of urea in 0.9% saline at a level of 0.8 mg. urea/g. body weight. The average weights of the animals used in these studies were as follows: alanine load: pregnant 325 g., nonpregnant 268 g.; casein hydrolyzate load: pregnant 310 g., nonpregnant 232 g.; urea load: pregnant 280 g., nonpregnant 210 g. The number of animals per group is indicated in each table. In the last experiment, the fetuses were removed from each uterus (6) on the 18th day of gestation; the placentas were left in situ. With another group of pregnant rats, the uterus was exposed and then replaced in the abdominal cavity; similar treatment was given to the group of nonpregnant rats. The average body weights of the groups at the time of operation were: pregnant 304 g., nonpregnant 281 g. Both the cesarotomized and control pregnant groups had an average of 8.3 fetuses/rat. The animals were killed on the 21st day of gestation following an 1%hr. fast. All animals were killed by stunning and decapitation. Blood was collected from the neck in heparinized tubes and assays were carried out by the following procedures: urea, Archibald (7); amino nitrogen, Frame et al. (8) as modified by Russell (9); packed cell volume by the standard procedure. In the last experiment, samples of liver were removed and immediately homogenized for the determination of transaminase activities by the following procedures: aspart,ic-glutamic, Tonhazy et al. (10) ; alanine-glutamic, Caldwell and McHenry (11). The statistical analysis of the results was carried out by means of the “t-test.” 1 Cod Liver Oil Concentrate, Ayerst, 50,000 I.U. of vitamin D and 200,000 I.U. * Wheat Germ Oil, VioBin (Canada) 3 Smigen, dried casein hydrolyzate; Company.
McKenna and Harrison Ltd.; containing of vitamin A/g. Ltd. provided gratis by Mead Johnson and
GRE.4 FORMATION IN PREGNAKT R.\T RESULTS
Administration
of DL-Alanine
In order to test in vivo urea formation, it was decided to esamine the blood urea levels following the injection of DL-alanine in pregnant and nonpregnant rats. Animals were killed at A-hr. intervals after treatment. The results are show1 in Table I. At 4 hr., the elevation of blood urea was significantly smaller in t.he pregnant group t,han in the nonpregnant animals (t = 7.76, significant at the 1% level). The levels were the same for both groups at 8 hr., and had returned to normal by 12 hr. Packed cell volumes indicated a slight hemoconcentration at 4 hr. followed b\ moderate hemodilution. The packed cell volumes of the pregnant rats were lower than those of the nonpregnant animals at all times, in agreement xvit.h previous observations (4). Visual examination of t.he abdominal cavity revealed complete absorption of the injected solution by 12 hr. The decreased urea response in pregnant rat,s was similar t.o that, rcported by McGanity ct al. (3) following the oral administration of nI,-alanine to pregnant women. hlministra.tion
of Casein Hydrolyzate
Since it is known (4) that pregnant rats exhibit a lowered alanineglutamic transaminase activity, it is possible that the blood urea response following the alanine load in pregnant rats was due to an impaired ability to utilize that particular amino acid. h mixture of amino acids in the form of casein hydrolyzate was given in a manner similar to the alanine; TABLE I Blood Urea Levels Following DL-Alanine Administration (Mean A standard error of the mean) Time after injection hr.
0 4 8 12
Group
No. of rats
Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant
6 5 4 8 5 7 5 7
Blood urea mg.%
35.7 33.9 57.7 99.2 57.7 56.4 31.9 36.3
i 2.2 f 2.5 * 3.9 f 3.2 f 5.3 f 1.8 f 1.3 f 3.1
GEORGE
H.
BEATON
TABLE II Observations Following the Administration o/ Casein Hydrolyzate 0Mean z!=standard error = of the mean) == =
-2%
injection
Blood urea
NO. of rats
Group
-
-
me%
‘1
Blood amino nitrogen _ 1 ._
Packed cell volume
hr.
0 2 4 6 12
Pregnant Nonpregnan Pregnant Nonpregnan Pregnant Nonpregnan Pregnant Nonpregnan Pregnant Nonpregnan
D Significant b Significant c Significant
8 33.8 8 33.2 11 77.3 t 7 88.9 11 86.5 t 9 116.0 9 89.6 t 7 121.0 10 36.7 t 11 63.8 == = at the 1% level. at the 2% level. at the 5yo level.
t
f 2.0 8.3 I10.1 i 1.7 f 2.5 2.73’ ‘1 .2.7 112.9 f 3.5 f 4.7 3.31‘ I 9.7 I11.3 f 4.1 i 9.1 2.91’ 5 9.3 111.1 f 4.6 1.1.7 2.22’ c 7.7 9.8 f 12.2 =
f * f f f f ff f f
0.35.81”42.6 0.2 50.2 0.2 - 48.1 0.6 58.0 0.33.62~45.6 0.3 55.3 0.43.62”44.7 0.3 56.9 0.28.91”37.9 0.1 44.9
f f zk f f * f f f f
2.03.64a 0.6 0.66.88* 1.6 1.05.350 1.7 1.75.19. 1.9 0.74.40” 1.5
thus, any difference in response could not be attributed to this one enzyme. Immediately following the injections, the rats appeared to enter a temporary state of shock, but recovered within 2 hr. Experimental results are shown in Table II. The mixture of amino acids gave the same type of urea response in the pregnant rats as did the single amino acid alanine. Again, initial hemoconcentration followed by hemodilution was seen; both groups showed approximately equal changes in blood volume; hence this was not responsible for the difference in urea response. Blood amino nitrogen levels reflect the initial rapid absorption of the injected amino acids and their subsequent removal from the blood stream. Although the pregnant rats exhibited lower amino nitrogen levels than did the controls, as has been reported previously (4), approximately parallel changes in the blood amino nitrogen were seenin both groups except for the greater initial rise in the pregnant rats. By 12 hr., amino nitrogen levels had returned to normal for both groups. Blood urea levels were normal in the pregnant rats at this time, but were still elevated in the nonpregnant rats.
UREA
FORMATION
IN
PREGNANT
5
RAT
Urea Administration. The previous experiments have demonstrated a difference between pregnant and nonpregnant rats in the blood urea response to injected amino acids. It is possible that this difference could be explained by a more rapid removal of urea from the blood of the pregnant rats than from that of the nonpregnant animals. The blood urea levels following the administration of urea itself are shown in Table III. The urea dosage was selected so as to produce blood urea levels approximating t.hose of the previous experiments. While the fasting blood urea levels were the same for both groups, the pregnant rats exhibited significantly higher levels than the controls at all t,imes after the administration of this compound. After 6 hr., the blood urea had returned to normal in the nonpregnant animals, but was still significantly above the initial level in t,he pregnant rats (t = 4.15, significant at the 1% level). Moderate hemodilution was produced to the same extent in both groups. From t.hese results, it appears that pregnant rats do not remove urea from the blood more rapidly than do controls; indeed, the results suggest that pregnant rats may exhibit a greater retention of urea. It is not likely that the higher blood urea TABLE Response of Pregnant Intraperitoneal (Mean Time after injection
f
III
and Nonpregna.nt Rats to an Injection of Urea
standard
error
-
T
Blood urea
-
-
Group
of the mean)
Packed cell volume
t
w.%
1
hr.
0 1 2 4 6
Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant
a Significant
s
i
at the 1% level.
9 8 10 8 10 8 9 8 9
-
31.9 30.7 105.9 92.6 92.3 76.7 63.8 49.3 52.6 29.0
f f f f f f f f f f
2.0 1.8 2.7 1.8 3.2 2.0 2.3 3.7 4.6 1.1
4.26a 4.34a 3.22. 5.320
41.8 49.6 39.8 50.4 41.7 48.2 34.9 46.2 35.3 47.2
f f f f f f zt f f f
1.2 0.7 0.8 1.5 1.3 0.9 0.6 1.4 0.7 0.7
5.60” 5.74a 4.36” 7.01= 12.3=
6
GEORGE H. BEATON
levels seen in the pregnant rats were due to the greater absolute dosage given. There was no visible correlation, within groups, between the amount of urea injected, or the body weight, and the blood urea response. Blood volume is proportional to body weight even in pregnant rats (12) ; hence the dilution of the injected urea should be approximately equal in all rats. It would appear then that pregnant rats actually form less urea following amino acid administration than do nonpregnant animals. Cesarotomy of Pregnant Rats
The lowered in viva formation of urea seen in pregnant rats could be due to one of two causes: (a) a rapid removal of amino acids from the blood stream in some manner other than urea formation, or (b) an actual decreasein the ability of the pregnant rat to form urea in agreement with in vitro studies (4). It could also be a combination of these two effects. Reports in the literature (13) suggest that the placenta can actively transport amino acids into the fetal circulation against a concentration gradient. Amino acids in the maternal blood are decreasedin pregnancy in the rat (4) and in the human (1, 13) ; fetal amino nitrogen levels are reported to be much higher than the maternal levels in the human (13) and in other species (14). This active withdrawal of amino acids may be the cause of the lowered maternal blood amino nitrogen during gestation. If this hypothesis is true, removal of the fetuses would be expected to return the maternal levels to the nonpregnant concentration. As can be seen in Table IV, cesarotomy, leaving the placentas in situ, had this expected effect on the blood amino nitrogen. The concentration of amino nitrogen in cesarotomized pregnant rats was almost identical with that of nonpregnant rats and much higher than that in shamoperated pregnant rats (t = 3.17, significant at the 1% level). The lowering of alanine-glutamic transaminase and packed cell volume normally seen in pregnancy (4) were not affected by cesarotomy, suggesting that at least some of the metabolic alterations of normal pregnancy were not disturbed. These results support the hypothesis that the cause of the low blood amino nitrogen in normal pregnancy is the withdrawal of amino acids from the maternal circulation into the fetal circulation for deposition in tissue protein. DISCUSSION
Following the administration of alanine or of casein hydrolyzate, pregnant rats showed a smaller elevation of blood urea than did nonpregnant
UREh
FORMATION
IN
PREGNi\NT
RST
TABLE IV The Effect of Cesarotomy on Pregnant Rats (Mean f standard error of the mean) Pregnant control
Assay
Transaminase activitya Alanine-glutamic Aspartic-glutamic Packed cell volume, ‘% Blood amino nitrogen, mg. Blood urea, mg. ?JO No. of rats a Activities tissue/hr.
To
21.5 104 41.3 7.6 31.8
f f f zk f 10
are expressed as microliters
1.6 3.2 0.8 0.3 1.5
Cesarotomized
pregnant
22.4 90 42.1 9.1 26.9
of pyruvate
f f f f f 8
1.4 1.3 1.2 0.2 2.1
NowEpt
42.1 98 47.9 9.3 26.4
i f f f f 9
CO1 formed/mg.
1.9 3.1 1.3 0.2 1.0 wet
controls. The amino acids were given in proportion to body weight and hence to blood volume; the difference in blood urea response could not have been due to unequal dilution of the injected amino acids. Within groups, no correlation was evident between the amount of amino acid injected (on a body-weight basis) and the urea response in the blood. Injected urea appeared to be removed more slowly from the blood of pregnant rats than was the case with nonpregnant controls. Previous studies have shown that the alanine-glutamic transaminase activity in the liver is reduced during pregnancy in the rat (4). There was no observed alteration in aspartic-glutamic transaminase activity. The change in the activity of the one transaminase might affect the production of urea from alanine but not from other amino acids. Previously reported results and those given in this paper indicate the following: (a) I?z Z&J experiments with liver slices showed that the rate of urea formation is reduced during pregnancy in the rat; (b) there is a smaller elevation of blood urea after the administration of amino acids to pregnant animals t#han was found in nonpregnant controls. These two conclusions are in agreement and both indicate a decrease in urea formation during gestation. Such an alteration should cause a lowering of fasting blood urea. The absence of such a change in pregnant rats is explicable by the finding that the urea clearance appears to be depressed. The decreasein urea removal would tend to keep the blood urea normal despite the diminution in urea formation. It will be recalled that it has been reported that fasting blood urea levels are not decreased during pregnancy in the rat (4). A decrease in blood amino nitrogen has been observed in pregnant
8
GEORGE H. BEATON
rats (4). The rise in blood amino nitrogen after surgical removal of the fetuses suggests that the depression seen in pregnant animals is due to a rapid withdrawal of amino acids through the placenta into the fetal circulation. Christensen and Streicher (13) have reported that the placenta can transfer amino acids against a concentration gradient. However, the blood amino nitrogen levels following t,he administration of casein hydrolyzate did not show a sufficiently rapid return to normal to indicate that the removal of amino acids to the fetal circulation could be the cause of the decreased urea formation. The in vitro studies showed a decreased rate of urea formation in liver tissue from pregnant rats (4). A similar reduction can be produced in nonpregnant rats by treatment wit’h anterior pituitary growth hormone (15). In vivo studies by Russell (16) also revealed a decreased urea formation following amino acid administration in growth-hormone-treated rats. It has already been pointed out that growth hormone treat#ment in nonpregnant rats can produce many of the met,abolic alterations seen in pregnancy (17) ; it was suggested that this hormone might play an important role in the control of metabolism in pregnancy. Recently, Contopoulos and Simpson (18) have reported elevated blood levels of this hormone in pregnant rats, thus giving support to this t,heory. In the nonpregnant rat, treatment with growth hormone results in an elevation of blood amino nitrogen; in the pregnant rat,, this would be counterbalanced by the rapid removal of amino acids to the fetus. Probably then, the decreased urea formation seen in pregnant rats is the result of a controlled alteration of protein metabolism. McGanity et al. (3) suggested that the decreased fasting blood urea seen in pregnant women was due to a decreased urea formation. This hypothesis was based on the blood urea response following the administration of alanine. The blood urea response reported by these workers was similar to that reported in this communication for pregnant rats after the administration of either alanine or casein hydrolyzate. McGanity et al. found that there was no change in the urea clearance of their patients. A decrease in urea production together with unaltered urea clearance explains the lowered blood urea in pregnancy in humans. It should be noted that the decrease in blood urea is greater than could be due to hemodilution. ACKNOWLEDGMENT This work was made possible by a Public Health Research Grant from the Department of National Health and Welfare of the Government of Canada.
URErl
FORiVATION
IN PREGNANT
RAT
9
SUMMARY
Pregnant rats exhibit a smaller elevation of blood urea following the intraperitoneal injection of either alanine or casein hydrolyzate than do nonpregnant controls. This is interpreted as further evidence of a decrease in urea formation in pregnancy. The pregnant rats showed a greater and more prolonged elevation of blood urea following the administration of urea t,han did nonpregnant controls; this could explain the lack of a decrease in the fasting blood urea during gestation in the rat. Data obtained from pregnant rats after the surgical removal of the fetuses suggest that the low blood amino nitrogen during pregnancy is clue to a withdrawal of amino acids into the fet,al circulation. REFERENCES 1. BEATON, J. R., CAVAN, M. J., LAU, R. E., h’kGmITY, W. J., MCHENRY, E. W., AND WATT, G. L., Bm. J. Obstet. Gyn.ecol. 62, 156 (1951). 2. NICE, M., J. Clin. Invest. 14, 575 (1935). 3. MCGANITY, W. J., MCHENRY, E. W., VANWYCK, H. B., AND WATT, G. L., J. Biol. Chem. 178, 511 (1949). 1. BEATON, G. H., BIZARE, J., RYU, M. H., AND MCHENRY, E. W., J. Nutrition 64, 291 (1954). 5. STEENBOCK, H., AND NELSON, E. M., J. Biol. Chem., 66, 362 (1923). 6. PRITCHARD, J. J., AND HUGGETT, 8. ST. G., J. Anat. 81,212 (1947). 7. ARCHIBALD, R. M., J. Biol. Chem. 167, 507 (1945). 8. FRAME, E. G., RUSSELL, J. A., AND WILHELMI, A. E., J. Biol. Chem. 149, 255 (1943). 9. RUSSELL, J. A., J. Biol. Chem. 166,467 (1944). 10. TONHAZY, N. E., WHITE, N. G., AND U~~MRREIT,W. W., Arch. Biochem. 28, 36 (1950). 11. CALDTVELL, E. F., AND MCHENRY, E. W., Arch. Biochem. and Biophys. 46, 97 (1953). 12. BOND, C. F., Endocrinology 43, 180 (1948). 13. CLEMETSON, C. A., J. Obstet. Gynaecol. Brit. Empire 61, 364 (1954). 14. CHRISTENSEN, H. N., AND STREICHER, J. A., J. Biol. Chem. 176,95 (1948). 15. BEATON, G. H., OZAWA, G., BEATON, J. R., AND MCHENRY, E. W., PTOC.Sot. Ezptl. Biol. Med. 83, 781 (1953). 16. RUSSELL, J. A., Endocrinology 49, 99 (1951). 17. BEATON, G. H., RYU, M. H., AND &HENRY, E. W., Endocrinology 67, 748 (1955). 18. CONTOPOULOS, A. N., AND SInwsoN, M. E., Federation Proc. 16, 39 (1956).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, l-9 (1957)
Urea Formation in the Pregnant Rat George H. Beaton From the Department of P-ublic Health Nutrition, University of Toronto, Toronto, Ontario
Received August 2, 1956
INTRODUCTION
A decreasein fasting blood urea is characteristic of human pregnancy While Nice (2) has stated that this is due to an increase in urea excretion, other workers (3) have found evidence of no change in urea clearance during pregnancy. In pregnant rats, the fasting blood urea either remains unchanged or is slightly increased. A decreased rate of urea production by slices of liver from pregnant rats has been found in uitro (4). It would be expected that the fetal demand for amino acids might be reflected by changes in amino acid catabolism in the maternal body. There could be a decreased formation of urea consequent to a lessened supply of amino acids for catabolism. This could explain the decreasedfasting blood urea of human pregnancy, but would not appear to be consistent with the unchanged blood urea in the pregnant rat. As part of a study of protein metabolism in pregnancy, it was decided to investigate urea formation, urea excretion, and the activities of several enzymes involved in urea formation. It has been possible to demonstrate that pregnant rats show a decreased formation and impaired excretion of urea. (1).
METHODS In all of the experiments to be described, female Wistar strain rats from t.he Carworth Farms colony were used. For breeding, the females were housed with males of the same strain, and vaginal smears were examined each morning; the day on which sperm was detected in the smear was taken as being day 0 of gestation. Following breeding, the pregnant and comparable nonpregnant control rats were housed in individual screen-bottomed cages in a room maintained at 75 f 2°F. and provided ad libitum with a 20yo corn oil, 20% casein diet. The diet con1
2
GEORGE
H.
BEATON
tained the following constituents in per cent by weight: casein 16, vitamin powder 4, corn oil 20, sucrose 54, nonnutritive cellulose (Alphacel) 2, salt mixture 4 [Steenbock-Nelson salts 140 (5)], choline 0.2, inositol 0.2, cod liver oil concentrate,’ 0.015. The vitamin powder was prepared by mixing thoroughly 800 g. casein with the following amounts (in milligrams) of vitamins: thiamine hydrochloride 100, riboflavine hydrochloride 100, pyridoxine hydrochloride 100, calcium pantothenate 400, nicotinic acid 400, p-aminobenzoic acid 400, biotin 20, folic acid 20. In the last experiment, vitamin E in the form of wheat germ oil* was added to the diet at a level of 10 mg./lOOO g. diet. The first three studies involved the intraperitoneal injection of various metabolites in pregnant and nonpregnant rats. In each case, the rats were fasted for 16-18 hr. prior to treatment. The initial control groups were injected with comparable volumes of 0.9% saline and killed immediately. The other groups were killed at the intervals indicated in the tables, following the injection of the metabolite. In the first experiment, on-alanine was administered as a 7.6% aqueous solution in an amount to provide 0.24 mg. nitrogen/g. body weight. Casein hydrolyaate’ was injected as an aqueous solution at a level of 0.36 mg. total nitrogen and 0.23 mg. amino nitrogen/g. of body weight. Both of these solutions were neutralized to pH 7.0 before injection. The third study involved the administration of a 4% solution of urea in 0.9% saline at a level of 0.8 mg. urea/g. body weight. The average weights of the animals used in these studies were as follows: alanine load: pregnant 325 g., nonpregnant 268 g.; casein hydrolyzate load: pregnant 310 g., nonpregnant 232 g.; urea load: pregnant 280 g., nonpregnant 210 g. The number of animals per group is indicated in each table. In the last experiment, the fetuses were removed from each uterus (6) on the 18th day of gestation; the placentas were left in situ. With another group of pregnant rats, the uterus was exposed and then replaced in the abdominal cavity; similar treatment was given to the group of nonpregnant rats. The average body weights of the groups at the time of operation were: pregnant 304 g., nonpregnant 281 g. Both the cesarotomized and control pregnant groups had an average of 8.3 fetuses/rat. The animals were killed on the 21st day of gestation following an 1%hr. fast. All animals were killed by stunning and decapitation. Blood was collected from the neck in heparinized tubes and assays were carried out by the following procedures: urea, Archibald (7); amino nitrogen, Frame et al. (8) as modified by Russell (9); packed cell volume by the standard procedure. In the last experiment, samples of liver were removed and immediately homogenized for the determination of transaminase activities by the following procedures: aspart,ic-glutamic, Tonhazy et al. (10) ; alanine-glutamic, Caldwell and McHenry (11). The statistical analysis of the results was carried out by means of the “t-test.” 1 Cod Liver Oil Concentrate, Ayerst, 50,000 I.U. of vitamin D and 200,000 I.U. * Wheat Germ Oil, VioBin (Canada) 3 Smigen, dried casein hydrolyzate; Company.
McKenna and Harrison Ltd.; containing of vitamin A/g. Ltd. provided gratis by Mead Johnson and
GRE.4 FORMATION IN PREGNAKT R.\T RESULTS
Administration
of DL-Alanine
In order to test in vivo urea formation, it was decided to esamine the blood urea levels following the injection of DL-alanine in pregnant and nonpregnant rats. Animals were killed at A-hr. intervals after treatment. The results are show1 in Table I. At 4 hr., the elevation of blood urea was significantly smaller in t.he pregnant group t,han in the nonpregnant animals (t = 7.76, significant at the 1% level). The levels were the same for both groups at 8 hr., and had returned to normal by 12 hr. Packed cell volumes indicated a slight hemoconcentration at 4 hr. followed b\ moderate hemodilution. The packed cell volumes of the pregnant rats were lower than those of the nonpregnant animals at all times, in agreement xvit.h previous observations (4). Visual examination of t.he abdominal cavity revealed complete absorption of the injected solution by 12 hr. The decreased urea response in pregnant rat,s was similar t.o that, rcported by McGanity ct al. (3) following the oral administration of nI,-alanine to pregnant women. hlministra.tion
of Casein Hydrolyzate
Since it is known (4) that pregnant rats exhibit a lowered alanineglutamic transaminase activity, it is possible that the blood urea response following the alanine load in pregnant rats was due to an impaired ability to utilize that particular amino acid. h mixture of amino acids in the form of casein hydrolyzate was given in a manner similar to the alanine; TABLE I Blood Urea Levels Following DL-Alanine Administration (Mean A standard error of the mean) Time after injection hr.
0 4 8 12
Group
No. of rats
Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant
6 5 4 8 5 7 5 7
Blood urea mg.%
35.7 33.9 57.7 99.2 57.7 56.4 31.9 36.3
i 2.2 f 2.5 * 3.9 f 3.2 f 5.3 f 1.8 f 1.3 f 3.1
GEORGE
H.
BEATON
TABLE II Observations Following the Administration o/ Casein Hydrolyzate 0Mean z!=standard error = of the mean) == =
-2%
injection
Blood urea
NO. of rats
Group
-
-
me%
‘1
Blood amino nitrogen _ 1 ._
Packed cell volume
hr.
0 2 4 6 12
Pregnant Nonpregnan Pregnant Nonpregnan Pregnant Nonpregnan Pregnant Nonpregnan Pregnant Nonpregnan
D Significant b Significant c Significant
8 33.8 8 33.2 11 77.3 t 7 88.9 11 86.5 t 9 116.0 9 89.6 t 7 121.0 10 36.7 t 11 63.8 == = at the 1% level. at the 2% level. at the 5yo level.
t
f 2.0 8.3 I10.1 i 1.7 f 2.5 2.73’ ‘1 .2.7 112.9 f 3.5 f 4.7 3.31‘ I 9.7 I11.3 f 4.1 i 9.1 2.91’ 5 9.3 111.1 f 4.6 1.1.7 2.22’ c 7.7 9.8 f 12.2 =
f * f f f f ff f f
0.35.81”42.6 0.2 50.2 0.2 - 48.1 0.6 58.0 0.33.62~45.6 0.3 55.3 0.43.62”44.7 0.3 56.9 0.28.91”37.9 0.1 44.9
f f zk f f * f f f f
2.03.64a 0.6 0.66.88* 1.6 1.05.350 1.7 1.75.19. 1.9 0.74.40” 1.5
thus, any difference in response could not be attributed to this one enzyme. Immediately following the injections, the rats appeared to enter a temporary state of shock, but recovered within 2 hr. Experimental results are shown in Table II. The mixture of amino acids gave the same type of urea response in the pregnant rats as did the single amino acid alanine. Again, initial hemoconcentration followed by hemodilution was seen; both groups showed approximately equal changes in blood volume; hence this was not responsible for the difference in urea response. Blood amino nitrogen levels reflect the initial rapid absorption of the injected amino acids and their subsequent removal from the blood stream. Although the pregnant rats exhibited lower amino nitrogen levels than did the controls, as has been reported previously (4), approximately parallel changes in the blood amino nitrogen were seenin both groups except for the greater initial rise in the pregnant rats. By 12 hr., amino nitrogen levels had returned to normal for both groups. Blood urea levels were normal in the pregnant rats at this time, but were still elevated in the nonpregnant rats.
UREA
FORMATION
IN
PREGNANT
5
RAT
Urea Administration. The previous experiments have demonstrated a difference between pregnant and nonpregnant rats in the blood urea response to injected amino acids. It is possible that this difference could be explained by a more rapid removal of urea from the blood of the pregnant rats than from that of the nonpregnant animals. The blood urea levels following the administration of urea itself are shown in Table III. The urea dosage was selected so as to produce blood urea levels approximating t.hose of the previous experiments. While the fasting blood urea levels were the same for both groups, the pregnant rats exhibited significantly higher levels than the controls at all t,imes after the administration of this compound. After 6 hr., the blood urea had returned to normal in the nonpregnant animals, but was still significantly above the initial level in t,he pregnant rats (t = 4.15, significant at the 1% level). Moderate hemodilution was produced to the same extent in both groups. From t.hese results, it appears that pregnant rats do not remove urea from the blood more rapidly than do controls; indeed, the results suggest that pregnant rats may exhibit a greater retention of urea. It is not likely that the higher blood urea TABLE Response of Pregnant Intraperitoneal (Mean Time after injection
f
III
and Nonpregna.nt Rats to an Injection of Urea
standard
error
-
T
Blood urea
-
-
Group
of the mean)
Packed cell volume
t
w.%
1
hr.
0 1 2 4 6
Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant Pregnant Nonpregnant
a Significant
s
i
at the 1% level.
9 8 10 8 10 8 9 8 9
-
31.9 30.7 105.9 92.6 92.3 76.7 63.8 49.3 52.6 29.0
f f f f f f f f f f
2.0 1.8 2.7 1.8 3.2 2.0 2.3 3.7 4.6 1.1
4.26a 4.34a 3.22. 5.320
41.8 49.6 39.8 50.4 41.7 48.2 34.9 46.2 35.3 47.2
f f f f f f zt f f f
1.2 0.7 0.8 1.5 1.3 0.9 0.6 1.4 0.7 0.7
5.60” 5.74a 4.36” 7.01= 12.3=
6
GEORGE H. BEATON
levels seen in the pregnant rats were due to the greater absolute dosage given. There was no visible correlation, within groups, between the amount of urea injected, or the body weight, and the blood urea response. Blood volume is proportional to body weight even in pregnant rats (12) ; hence the dilution of the injected urea should be approximately equal in all rats. It would appear then that pregnant rats actually form less urea following amino acid administration than do nonpregnant animals. Cesarotomy of Pregnant Rats
The lowered in viva formation of urea seen in pregnant rats could be due to one of two causes: (a) a rapid removal of amino acids from the blood stream in some manner other than urea formation, or (b) an actual decreasein the ability of the pregnant rat to form urea in agreement with in vitro studies (4). It could also be a combination of these two effects. Reports in the literature (13) suggest that the placenta can actively transport amino acids into the fetal circulation against a concentration gradient. Amino acids in the maternal blood are decreasedin pregnancy in the rat (4) and in the human (1, 13) ; fetal amino nitrogen levels are reported to be much higher than the maternal levels in the human (13) and in other species (14). This active withdrawal of amino acids may be the cause of the lowered maternal blood amino nitrogen during gestation. If this hypothesis is true, removal of the fetuses would be expected to return the maternal levels to the nonpregnant concentration. As can be seen in Table IV, cesarotomy, leaving the placentas in situ, had this expected effect on the blood amino nitrogen. The concentration of amino nitrogen in cesarotomized pregnant rats was almost identical with that of nonpregnant rats and much higher than that in shamoperated pregnant rats (t = 3.17, significant at the 1% level). The lowering of alanine-glutamic transaminase and packed cell volume normally seen in pregnancy (4) were not affected by cesarotomy, suggesting that at least some of the metabolic alterations of normal pregnancy were not disturbed. These results support the hypothesis that the cause of the low blood amino nitrogen in normal pregnancy is the withdrawal of amino acids from the maternal circulation into the fetal circulation for deposition in tissue protein. DISCUSSION
Following the administration of alanine or of casein hydrolyzate, pregnant rats showed a smaller elevation of blood urea than did nonpregnant
UREh
FORMATION
IN
PREGNi\NT
RST
TABLE IV The Effect of Cesarotomy on Pregnant Rats (Mean f standard error of the mean) Pregnant control
Assay
Transaminase activitya Alanine-glutamic Aspartic-glutamic Packed cell volume, ‘% Blood amino nitrogen, mg. Blood urea, mg. ?JO No. of rats a Activities tissue/hr.
To
21.5 104 41.3 7.6 31.8
f f f zk f 10
are expressed as microliters
1.6 3.2 0.8 0.3 1.5
Cesarotomized
pregnant
22.4 90 42.1 9.1 26.9
of pyruvate
f f f f f 8
1.4 1.3 1.2 0.2 2.1
NowEpt
42.1 98 47.9 9.3 26.4
i f f f f 9
CO1 formed/mg.
1.9 3.1 1.3 0.2 1.0 wet
controls. The amino acids were given in proportion to body weight and hence to blood volume; the difference in blood urea response could not have been due to unequal dilution of the injected amino acids. Within groups, no correlation was evident between the amount of amino acid injected (on a body-weight basis) and the urea response in the blood. Injected urea appeared to be removed more slowly from the blood of pregnant rats than was the case with nonpregnant controls. Previous studies have shown that the alanine-glutamic transaminase activity in the liver is reduced during pregnancy in the rat (4). There was no observed alteration in aspartic-glutamic transaminase activity. The change in the activity of the one transaminase might affect the production of urea from alanine but not from other amino acids. Previously reported results and those given in this paper indicate the following: (a) I?z Z&J experiments with liver slices showed that the rate of urea formation is reduced during pregnancy in the rat; (b) there is a smaller elevation of blood urea after the administration of amino acids to pregnant animals t#han was found in nonpregnant controls. These two conclusions are in agreement and both indicate a decrease in urea formation during gestation. Such an alteration should cause a lowering of fasting blood urea. The absence of such a change in pregnant rats is explicable by the finding that the urea clearance appears to be depressed. The decreasein urea removal would tend to keep the blood urea normal despite the diminution in urea formation. It will be recalled that it has been reported that fasting blood urea levels are not decreased during pregnancy in the rat (4). A decrease in blood amino nitrogen has been observed in pregnant
8
GEORGE H. BEATON
rats (4). The rise in blood amino nitrogen after surgical removal of the fetuses suggests that the depression seen in pregnant animals is due to a rapid withdrawal of amino acids through the placenta into the fetal circulation. Christensen and Streicher (13) have reported that the placenta can transfer amino acids against a concentration gradient. However, the blood amino nitrogen levels following t,he administration of casein hydrolyzate did not show a sufficiently rapid return to normal to indicate that the removal of amino acids to the fetal circulation could be the cause of the decreased urea formation. The in vitro studies showed a decreased rate of urea formation in liver tissue from pregnant rats (4). A similar reduction can be produced in nonpregnant rats by treatment wit’h anterior pituitary growth hormone (15). In vivo studies by Russell (16) also revealed a decreased urea formation following amino acid administration in growth-hormone-treated rats. It has already been pointed out that growth hormone treat#ment in nonpregnant rats can produce many of the met,abolic alterations seen in pregnancy (17) ; it was suggested that this hormone might play an important role in the control of metabolism in pregnancy. Recently, Contopoulos and Simpson (18) have reported elevated blood levels of this hormone in pregnant rats, thus giving support to this t,heory. In the nonpregnant rat, treatment with growth hormone results in an elevation of blood amino nitrogen; in the pregnant rat,, this would be counterbalanced by the rapid removal of amino acids to the fetus. Probably then, the decreased urea formation seen in pregnant rats is the result of a controlled alteration of protein metabolism. McGanity et al. (3) suggested that the decreased fasting blood urea seen in pregnant women was due to a decreased urea formation. This hypothesis was based on the blood urea response following the administration of alanine. The blood urea response reported by these workers was similar to that reported in this communication for pregnant rats after the administration of either alanine or casein hydrolyzate. McGanity et al. found that there was no change in the urea clearance of their patients. A decrease in urea production together with unaltered urea clearance explains the lowered blood urea in pregnancy in humans. It should be noted that the decrease in blood urea is greater than could be due to hemodilution. ACKNOWLEDGMENT This work was made possible by a Public Health Research Grant from the Department of National Health and Welfare of the Government of Canada.
URErl
FORiVATION
IN PREGNANT
RAT
9
SUMMARY
Pregnant rats exhibit a smaller elevation of blood urea following the intraperitoneal injection of either alanine or casein hydrolyzate than do nonpregnant controls. This is interpreted as further evidence of a decrease in urea formation in pregnancy. The pregnant rats showed a greater and more prolonged elevation of blood urea following the administration of urea t,han did nonpregnant controls; this could explain the lack of a decrease in the fasting blood urea during gestation in the rat. Data obtained from pregnant rats after the surgical removal of the fetuses suggest that the low blood amino nitrogen during pregnancy is clue to a withdrawal of amino acids into the fet,al circulation. REFERENCES 1. BEATON, J. R., CAVAN, M. J., LAU, R. E., h’kGmITY, W. J., MCHENRY, E. W., AND WATT, G. L., Bm. J. Obstet. Gyn.ecol. 62, 156 (1951). 2. NICE, M., J. Clin. Invest. 14, 575 (1935). 3. MCGANITY, W. J., MCHENRY, E. W., VANWYCK, H. B., AND WATT, G. L., J. Biol. Chem. 178, 511 (1949). 1. BEATON, G. H., BIZARE, J., RYU, M. H., AND MCHENRY, E. W., J. Nutrition 64, 291 (1954). 5. STEENBOCK, H., AND NELSON, E. M., J. Biol. Chem., 66, 362 (1923). 6. PRITCHARD, J. J., AND HUGGETT, 8. ST. G., J. Anat. 81,212 (1947). 7. ARCHIBALD, R. M., J. Biol. Chem. 167, 507 (1945). 8. FRAME, E. G., RUSSELL, J. A., AND WILHELMI, A. E., J. Biol. Chem. 149, 255 (1943). 9. RUSSELL, J. A., J. Biol. Chem. 166,467 (1944). 10. TONHAZY, N. E., WHITE, N. G., AND U~~MRREIT,W. W., Arch. Biochem. 28, 36 (1950). 11. CALDTVELL, E. F., AND MCHENRY, E. W., Arch. Biochem. and Biophys. 46, 97 (1953). 12. BOND, C. F., Endocrinology 43, 180 (1948). 13. CLEMETSON, C. A., J. Obstet. Gynaecol. Brit. Empire 61, 364 (1954). 14. CHRISTENSEN, H. N., AND STREICHER, J. A., J. Biol. Chem. 176,95 (1948). 15. BEATON, G. H., OZAWA, G., BEATON, J. R., AND MCHENRY, E. W., PTOC.Sot. Ezptl. Biol. Med. 83, 781 (1953). 16. RUSSELL, J. A., Endocrinology 49, 99 (1951). 17. BEATON, G. H., RYU, M. H., AND &HENRY, E. W., Endocrinology 67, 748 (1955). 18. CONTOPOULOS, A. N., AND SInwsoN, M. E., Federation Proc. 16, 39 (1956).