We have seen that some amino acids are utilized by the animal organism as detoxication agents and that products like benzoyl-glycine (hippuric acid) or phenyl-acetyl-glutamine can be found in urine. We will also see that urobilin and stercobilin, the degradation products of hemoglobin (see fig. 7-33), are present in faeces.
But generally, nitrogen is eliminated in 3 forms:
1. Ammonia and ammonium salts in ammoniotelic organisms (aquatic invertebrates, fishes);
2. Uric acid, in uricotelic species (terrestrial invertebrates, birds, reptiles);
3. Urea in ureotelic animals (especially mammals including man).
These various nitrogeneous wastes can co-exist in the same organism; for example, man excretes nitrogen mostly in the form of urea (80%), but ammonium salts and uric acid are also found in urine.
Ammonia is a highly toxic product; the organism must be able to eliminate it fast and that is probably why this form of elimination was maintained in aquatic animals where NH3 can be expelled rapidly by diffusion into the external medium.
In terrestrial animals less toxic products are used: uric acid and urea. This idea is confirmed by the fact that some animals (e.g., the tadpole) excrete their nitrogen in the form of NH3 during the aquatic period of their life, but acquire the property of forming urea when they leave water (frog).
1. Ammonia and Ammonium Salts:
As observed above, in most tissues, the deamination of amino acids leads to the formation of NH3. This toxic ammonia is taken over by glutamic acid to form glutamine and it is in this form that NH3 is transported by blood up to the kidneys.
The glutamic acid-glutamine system therefore plays a capital role in the transport of NH3, and it must be mentioned that these 2 amino acids represent a very high percentage of the non-protein nitrogen of animal tissues (in human plasma they together form 1/3 of the total nitrogen of amino acids).
In the kidneys, glutaminase catalyzes the reverse reaction and decomposes glutamine which thus provides the major part of urine NH3. The rest is produced by the deamination of amino acids in the renal parenchyma. In the liver, the ammonia produced by glutamate dehydrogenase is directly detoxified in the urea cycle by a mechanism we will study in the next paragraph.
In the case of acidosis, for example during prolonged physical exercise (production of lactic acid), or in diabetes (ketonemia), or after injection of acid to an animal, excretion of NH3 increases – at the cost of urea formation — because it is used for neutralizing acidity, which permits an economy of other ions required by the organism (Na+, Ca2+, Mg2+); therefore, in acidosis, more NH3 is eliminated and less urea.
Exactly the reverse takes place in the case of alkalosis. It may be said that in general, in an animal receiving a fixed diet, the total nitrogen excreted ( NNH3 + Nurea) does not vary much, but the respective proportions of the two forms of elimination can vary.
As we will see now, NH3 is a precursor of urea, but it must remembered that the reverse is not true (urea is not a precursor of urine NH3, the precursor is mostly blood glutamine, and — to a lesser extent — all the amino acids whose deamination in the kidney produces NH3 which is immediately excreted).
2. Ureogenesis:
Urea formation is localized in the liver; this was shown by various observations and experiments and especially the fact that if an isolated liver is perfused with a solution containing NH3, the liquid flowing out is free from NH3 but contains urea. Krebs and Henseleit succeeded in bringing about urea formation by slices of rat liver and showed that arginase catalyzes urea formation from arginine.
But there was not enough free arginine to explain urea production by the hydrolysis of this amino acid, and this led to the idea that arginine acts catalytically; it was later found that two other amino acids, ornithine and citrulline have the same action and Krebs proposed the mechanism represented in figure 7-30, known as ornithine cycle, urea cycle, or Krebs-Henseleit cycle, the various steps of which we will examine now.
A. Synthesis of Carbamyl-Phosphate and Transformation of Ornithine into Citrulline:
The formation of carbamyl phosphate is catalyzed by a mitochondrial enzyme using NH3 as substrate (the latter results from the deamination of glutamate by glutamate dehydrogenase).
A derivative of glutamate, acetyl- glutamate, is the allosteric activator of this carbamyl-phosphate synthetase (the carbamyl-phosphate required for the synthesis of pyrimidines is synthesized by a cytosolic enzyme, using glutamine as substrate and being regulated in a totally different manner).
Carbamyl-phosphate reacts with the δ-amino group of ornithine to give citrulline, under the influence of ornithine-carbamyl-transferase. It must be noted that during this step, carbamyl phosphate supplies the carbon atom and one of the nitrogen atoms of the future urea molecule.
B. Transformation of Citrulline into Arginine:
This transformation requires two reactions. In a first step, citrulline condenses with aspartic acid in presence of ATP to form arginosuccinic acid, which, in a second step is split into arginine and fumaric acid, it may be observed that the aspartic acid supplies the second nitrogen atom of the future urea molecule.
This transfer in two steps of the amino group of aspartic acid (which is converted into fumaric acid in the process) is quite similar to the transfers we studied, on one hand during the input of N, in the biosynthesis of the purine ring, and on the other hand during the amination of IMP to AMP.
The immediate donor of NH2 is therefore obligatorily aspartic acid, but we have seen that the amino group of aspartic acid may result from glutamic acid (by transamination to oxaloacetic acid) and that the amino group of glutamic acid may itself result from a large number of amino acids since glutamic acid play a very active part in transamination processes. The 2NH2 of the urea molecule may therefore originate from various amino acids.
C. Transformation of Arginine into Ornithine and Urea:
This hydrolysis is catalyzed by arginase and leads to urea on one hand and ornithine on the other; ornithine is thus regenerated and can begin a new turn of the cycle by binding a new carbamyl-phosphate molecule.
D. Relations between the Cycle of Urea and the Cycle of Tricarboxylic Acids:
It is observed that the amino group of amino acids can be eliminated in the form of urea through 2 pathways which join in the ornithine cycle: either by deamination and then incorporation in carbamyl-phosphate, or by transamination to aspartic acid (with a possible intermediate passage through glutamic acid).
In the latter case, it is apparent that there is a close relationship between the urea cycle and the cycle of tricarboxylic acids, thanks to aspartic acid and fumaric acid which establish the link between the 2 cycles: the oxaloacetic acid of the cycle of tricarboxylic acids can be transaminated, and the aspartic acid thus formed enters the urea cycle; the fumaric acid thus formed returns to the cycle of tricarboxylic acids.
Furthermore, transamination which gives rise to aspartic acid often takes place at the cost of glutamic acid, which is itself formed from α-ketoglutaric acid, another intermediate of the cycle of tricarboxylic acids (see fig. 7-31).
Besides, energetic relations exist between the 2 cycles since the synthesis of urea requires ATP for the synthesis of carbamyl-phosphate and also for the condensation of citrulline and aspartic acid, and the ATP required can be supplied by the phosphorylations which accompany the oxidation of the intermediates of the cycle of tricarboxylic acids. Krebs and Henselheit had in fact observed that the synthesis of urea in their experiments necessitated the presence of oxygen and the addition of an oxidizable substrate.
3. Uric Acid:
In uricotelic animals like birds, uric acid is the principal form of elimination of nitrogen; it is a compound possessing the purine ring which is synthesized from various precursors. These animals therefore perform a series of relatively complex reactions to eliminate their nitrogen.
In man, uric acid is also found in urine; it results from the catabolism of purine nucleotides (which are themselves produced by the hydrolysis of nucleic acids). Figure 7-32 shows the catabolism of the two purine bases, adenine and guanine, which appear after the action of nucleosidases on nucleosides (themselves formed by action of nucleotidases on nucleotides).
It is observed that the two bases are first deaminated, then oxidized thanks to an enzyme called xanthine-oxidase which catalyzes the transformation of xanthine into uric acid and functions with a flavin coenzyme transferring the hydrogen atoms directly to the oxygen.
In man, the catabolism of the purine ring ends there, uric acid is eliminated in urine. In a disease called gout, uric acid salts (urates) of low solubility, are deposited in the joints.
But in most animals the catabolism of the purine ring continues (see fig. 7-32) up to the allantoin stage (in many mammals), the allantoic acid stage (in some fishes), or even up to the urea and glyoxylic acid stage. Lastly, let us mention that some bacteria possess an enzyme, urease, which can hydroiyze urea to CO2 and NH3.
4. Catabolism of Porphyrins:
The porphyrin group of hemoglobin remains stable for the entire life of the erythrocyte (about 4 months), as maybe noted by administering the 15N isotope whose level in the prosthetic group remains constant during this period.
Then, hemoglobin is degraded mostly in the spleen — up to the bilirubin stage (see fig. 7-33); bilirubin is then transported to the liver where it is conjugated with glucuronic acid, before being eliminated through the bile, into the intestine.
When elimination of the bile is prevented by an obstacle (e.g., a calculus), the bile constituents flow back into the blood, particularly the glucorono-conjugated derivative of bilirubin, which can then pass into the urine. But when bile flows normally into the intestine, bilirubin reaches there and undergoes a new series of transformations under the influence of intestinal bacteria and their enzymes.
Free bilirubin is first regenerated by hydrolysis of the glucorono-conjugate and then the following may take place (see fig. 7-33):
1. Reduction of the 2 vinyl groups (on pyrrol I and pyrrol II) to ethyl groups, which gives mesobilirubin;
2. Reduction of the 2 β and δ — CH2= bridges to — CH3 —, which gives mesobilirubinogen (or urobilinogen).
There are then 2 possibilities:
1. Either dehydrogenation of the γ methylene bridge and the nitrogen of pyrrol IV, which gives urobilin,
2. Or reduction of the double bonds of pyrrol I and pyroll II, which gives stercobilinogen which is transformed into stercobilin by a dehydrogenation of the γ methylene bridge and the nitrogen of pyrrol IV.
Urobilin and stercobilin are excreted in the faeces which take up their coloration (this is why stools are sometimes discoloured during disorders of bile secretion). As for uribilinogen and stercobilinogen, they are partly re-absorbed by the intestinal mucosa and return to the liver; an entero-hepatic cycle of bile pigments is thus produced.