ABSTRACT
An analysis, more extensive than hitherto, has been made of nitrogen excretion in developing chicks. It was found that the concentration of ammonia in the allantoic fluid was constant from the 5th to the 11th day and equalled the concentration in the extra-allantoic fluids, e.g. yolk. The increase into total ammonia content of the allantois during development is apparently due to the increase in volume of the organ and not to excretion by the embryo.
The concentration of urea in the allantoic fluid increases during development and injected urea concentrates in that organ.
The total amount and concentration of uric acid in allantoic fluid increases during development. The ratio of urea nitrogen to uric-acid nitrogen is relatively constant from the 6th to the 11th day of development indicating that no major shift from urea excretion to uric-acid excretion occurs.
No urease activity was observed even in the blastodiscs of unincubated eggs.
The distribution of arginase activity in 7-day-old chicks did not resemble that of ureotelic organisms. Activity of heads, bodies, and livers were essentially the same.
From these results it was concluded that ammonia is not an excretion product and that the developing chick does not pass through either a typical ammonotelic or ureotelic stage of excretion.
INTRODUCTION
OF the many examples of metabolic changes occurring during development, the most widely cited is that in developing chicks wherein the end product of nitrogen metabolism has been assumed to shift from ammonia to urea and finally to uric acid (Needham, 1931). This system appeared to be ideal for a proposed study of the mechanisms bringing about such changes in metabolism, providing the occurrence of these changes could be further substantiated. The experimental evidence upon which this pattern had been postulated consisted entirely of measurements of the total amounts of ammonia, urea, and uric acid in the allantois and the ratio of their total weights to the weight of the embryo. Nothing was reported concerning the changes in concentration of these substances nor their presence outside the allantois. Lack of this information makes it impossible to conclude that net synthesis occurs, particularly in the cases of ammonia and urea where the total amounts present are quite small.
If such a series of changes occurs in the excretion pattern of the developing chick, it is necessary that the enzymatic patterns of the organism undergo a concurrent series of transformations. In this connexion there appears in the literature only an incomplete analysis of arginase activity in developing chicks (Needham & Brachet, 1935).
In this paper there is presented an analysis of the nitrogenous contents of the allantois and the entire egg, a study of the fates of injected nitrogenous substances, and an analysis of embryos for the enzymes urease and arginase. These investigations have yielded evidence which we believe clearly shows that ammonia is not an excretion product and that the embryo does not pass through stages of ammonia or urea excretion characteristic of its evolutionary ancestry.
MATERIALS AND METHODS
Eggs used for these studies came from a flock of white leghorns and were incubated at 39° C. in a commercial incubator. Eggs were injected by placing 0·5 ml. of either distilled water or the appropriate solution in the air-sac. Allantoic fluid was removed with a hypodermic syringe through a hole in the blunt end of the egg.
Chemical determinations
Ammonia was determined by the method of Boyce (1950) substituting Dow-Corning antifoam A for the antifoam agent used in the original procedure. This method involves the removal of ammonia from complex mixtures by passing air through an alkaline aliquot into 0·1 N H2SO4 which traps the volatile ammonia. The quantity present is then determined by the use of Nesaler’s Reagent according to the method of Folin & Wu (1919). All analyses were run in duplicate.
Urea was determined by treating the sample with a standard solution of jack bean meal urease (Folin & Wu, 1919) and measuring the resulting ammonia by the method described above.
Uric acid in the allantoic fluid was determined by the glycerol-silicate method of Forsham et al. (1948).
Total nitrogen was determined by a modified Kjeldahl digestion (Ma & Zuazaga, 1942) followed by nesalerization according to the procedure used for ammonia determinations.
Enzyme determinations
Urease was measured by the method of Van Slyke & Cullen (1914) wherein a tissue homogenate is incubated with a standard buffered (pH 7·0) solution of urea, at room temperature for various time intervals. After stopping the enzymatic activity by addition of acid the amount of ammonia formed is determined as described above.
Arginase activity was determined according to the procedure of Van Slyke & Archibald (1946) as follows: (1) tissues are homogenized in an appropriate amount of distilled water; (2) 0·25 ml. of a 20 per cent, solution of MnC12.4H2O is added to a 5 ml. aliquot of the homogenate; (3) the mixture is incubated for 20 minutes at 56° C. (to activate the enzyme); (4) after cooling, 2 · 5 ml. of an 18 per cent, solution of arginine (adjusted to pH 9·5) is added; (5) the reaction mixture is incubated at room temperature (approximately 25° C.); and (6) 1-ml. samples are removed after 0,20,40, and 60 minutes of incubation and placed in test-tubes maintained for 10 minutes at 90° C. in a water-bath to stop enzymatic activity. The 1-ml. samples are adjusted to pH 7 with 0 · 2 M phosphate buffer; the urea formed is hydrolysed with urease; the resulting ammonia is determined as described above.
RESULTS AND DISCUSSION
Ammonia excretion
Needham (1926a, 1931) has published data showing that the total amount of ammonia in the allantois increases during chick development from 3μg. at 4 days to 56 at 13 days, and has shown that the ratio of allantoic ammonia to weight of embryo decreases during this entire period. From these facts he postulated that chicks excrete ammonia in early stages and gradually lose this capacity with urea becoming the predominant excretory product.
Our data are in agreement with his experimental findings that the total amount of ammonia in the allantois increases during development; however, measurements (Table 1) show that the concentration remains constant. The concentration of ammonia in the whole egg contents (homogenized) was found to equal that in the allantois, and the total amount in the developing egg remained relatively constant from the 1st through the 13th day (Table 1). Samples were pooled from a number of allantoic sacs (e.g. 13 on 5th day, 3 on 11th day) to obtain adequate volumes for analysis. The volumes of allantoic sacs were determined from a standard plot made by removing the fluid contents with a syringe from ten replicate eggs at each day and measuring the volume obtained. These values agreed very closely with those found by Fiske & Boyden (1926).
When 1 mg. of ammonia nitrogen (as a buffered ammonium sulphate solution) is placed in the air-sac of eggs which have been incubated for 6 days, it becomes equally distributed between the yolk and allantois raising the concentration of ammonia in each threefold. This excess ammonia disappears rapidly, the value for both yolk and allantois becoming equal to the controls injected with distilled water within 2 days (Table 2).
If the early chick embryo, from the 1st to the 5th day, is an ammonia-excreting organism, it is necessary that urease be present, otherwise purine nitrogen would not be eliminated as ammonia. All attempts to demonstrate urease activity have been unsuccessful, even in the blastodiscs of unincubated eggs. The methods used were so sensitive that the production of 1μg. of ammonia nitrogen by an entire embryo in 1 hour would have been detected.
These data show that (1) the total amount of ammonia in the allantois increases during development; (2) the concentration of ammonia in the allantois remains constant from the 5th through at least the 11th day; (3) throughout this period the concentration of ammonia outside the allantois equals that inside; (4) ammonia injected as ammonium sulphate into a developing egg rapidly becomes distributed uniformly in the yolk and allantois, increasing the concentrations in each to the same extent; (5) the excess ammonia resulting from the injection disappears from both the allantois and the yolk in less than 2 days’ time; and (6) no urease activity is detectable from as early as the blastoderm stage.
The existing evidence does not warrant any conclusion concerning the presence of an active ammonia excreting mechanism during the first 5 days of development, and that after the 5th day there is no net excretion of ammonia into the allantoic sac.
Urea excretion
As in the case of ammonia, Needham (1926b, 1931) has reported data showing (1) that the total amount of urea in the allantois increases during chick development; (2) that the ratio of urea to the weight of the embryo increases from the 4th to the 8th day; and (3) that this ratio decreases from the 8th day to the time of hatching. He has postulated from these facts that the chick passes through a stage of high urea excretion.
Our analyses are in agreement that the total amount of urea in the allantois increases during development (Table 3). It was also found (1) that, unlike ammonia, the allantoic concentration of urea does not remain constant but increases from the 5th through at least the 11th day; (2) that the total amount of urea in the entire egg contents increases during the entire period studied; and (3) that the concentration of urea in the allantoic fluid is several fold higher than in the surrounding fluids (by the 11th day the allantois contains one-half of the urea present inside the entire egg, see Table 3).
When 1 mg. of urea nitrogen (as a urea solution) is placed in the air-sacs of eggs, which have been incubated for 6 days, it rapidly becomes concentrated in the allantoic sac and remains there (Table 4). By the 11th day 40 per cent, of all the injected urea has been concentrated in the allantois even though the sac occupies only 10 per cent, of the total volume.
If the developing chick embryo passes through a stage wherein urea is the primary excretion product and shifts to a stage wherein uric acid is the primary product, one should expect to find a shift in the relative amounts of each being excreted. The daily increases in concentration of urea in the allantoic fluid from the earliest time measured (5th day) through the 11th were approximately 1 (?), 5, 5,5, 3, and 2 μg. of urea N/ml. (see Table 3); comparable values for uric acid were 30,36,32,46,28, and 31 μg. uric acid N/ml. (see Table 6). Hence, from the earliest times at which measurement could be made the relative amounts of each excreted were reasonably constant and uric acid is the major excretory product as early as the 5th day.
The presence of a ureotelic metabolism in a developing chick would require that the enzyme arginase be present in order for protein nitrogen to be eliminated. It has been shown (Needham & Brachet, 1935) that the arginase activity per milligramme moist weight of developing chick embryos decreases during development from the 3rd to the 15th day. If the arginase in the early chick embryo is the result of a recapitulation of a ureotelic excretory metabolism certain organs (primarily the liver) should possess a higher concentration of the enzyme than others (Hunter & Dauphinee, 1924).
We have measured the arginase activity of intact embryos from 2·5 to 15 days, of separated heads and bodies from the 7th to the 15th day, and of isolated livers of 7-day-old chick embryos. No differences in activity were found (Table 5). On the basis of these data it is concluded that during the mesonephric stage of development chicks do not recapitulate the arginase distribution associated with a typical ureotelic physiology.
These studies show: (1) that the total amount of urea in the allantois increases during development; (2) that the concentration of urea in the allantoic sac also increases from the 5th through at least the 11th day; (3) that the concentration of urea is greater inside the allantois than outside; (4) that the injected urea becomes concentrated in the allantois; (5) the fraction of nitrogen excreted as urea remains relatively constant from the 6th through at least the 11th day; and (6) that arginase is not distributed among the various tissues of the developing chick in a manner characteristic of ureotelic organisms.
It is concluded that the developing chick excretes urea, but that the rate of urea excretion does not reach a maximum at any particular stage, and that the organism does not recapitulate a typical ureotelic excretion physiology.
Uric acid excretion
Although uric acid excretion has been extensively studied by many investigators (a list has been compiled by Needham, 1931), it was included in our studies for purposes of obtaining a more complete picture (Table 6).
When half a milligramme of uric acid nitrogen (as a sodium ureate solution adjusted to pH 7) was placed in the air-sac of eggs which have been incubated for 6 days, it gradually accumulated in the allantoic sac until essentially all of it appeared there by the 11th day (Table 6).
By dividing the amount of uric acid by the weight of the embryo, Needham (1926ft, 1931) obtained an excretion curve which is essentially zero from the 4th to the 7th day, which raises to a peak at the 11th day and then decreases. This curve can be interpreted as meaning that the embryo changes from a urea-excreting organism to an essentially uric-acid-excreting organism during the period from the 7th to the 11th day. If the change were as dramatic as would appear from this particular method of analysis of the uric-acid data, one should expect to find a shift in the relative amounts of each being excreted. As was shown previously (section on urea excretion) the daily increments in concentration in the allantois due to urea and uric acid are essentially constant from the 5th through at least the 11th day, and throughout this entire period the value for urea is only about one-sixth that for uric acid. Hence, from the earliest times at which measurements could be made the relative amounts of each excreted were reasonably constant and it is concluded that uric acid is the major excretory product as early as the 5th day.
Nitrogenous composition of allantoic fluid in developing chicks
To conclude this study of nitrogen excretion in developing chicks the total nitrogen in the allantois was determined to find out how much of the nitrogen present was accounted for by ammonia, urea, and uric acid.
Our analyses leave about 60 per cent, of the nitrogen to be accounted for. This is considerably more than calculated by Needham (1931) from data compiled from several investigators. Our unassignable fraction is larger than his value primarily because our uric-acid values were lower than those he used (Fiske & Boyden, 1926); however, our values are actually higher than Needham’s (1926) own experimental values.
CONCLUSIONS
Ammonia excretion
Because (1) the concentration of ammonia inside and outside the allantois are constant and equal from the 5th through at least the 11th day; (2) ammonia injected into the egg moves rapidly and equally into the yolk and allantois (rapidly disappearing from both); and (3) no urease activity was detected, it is concluded that ammonia is not an excretion product after the 5th day of development, that it need not necessarily be considered a product of active excretion prior to this time, and that the increase in total ammonia in the allantois is due only to the increasing volume of that organ.
Urea excretion
Because (1) the concentration of urea inside the allantois is greater than that outside and increases during development; (2) urea injected into the egg moves rapidly into the allantois and remains there; (3) the increments of urea concentration in the allantois are essentially constant from the 5th to the 11th day; and (4) arginase is uniformly distributed among the various tissues of the embryo, it is concluded that although urea is an excretion product from the 5 th through at least the 11th day, no predominantly ureotelic stage exists during chick development.
Uric acid excretion
Because (1) the concentration and total amount of uric acid in the allantois increases from the 5th to at least the 11th day; (2) uric acid injected into the egg accumulates in the allantois; and (3) the daily increments of uric-acid concentration in the allantois are equal from the 5th through at least the 11th day, it is concluded that uric acid is a major excretion product from the 6th to the 11th day and that no marked shift from urea to uric-acid excretion occurs during this period.
Total nitrogen in the allantois
The total amount of nitrogen was determined and 40 per cent, was accounted for as ammonia, urea, or uric acid.