Catabolites and Excretion

Carbon dioxide is a gas resulting from decarboxylation reactions and an important ubiquitary source of the cat-abolite is the Krebs cycle. Carbon dioxide exists in solution in equilibrium in its hydrated form, namely carbonic acid:

CO2 + H2O ^ H2CO3 ^ H+ + HCO3- ^ H+ + CO32-, Keq — 103, pKa1 — 6.1 (calculated with respect to dissolved CO2), pKa2 — 10.3at 25 °C

Carbonic acid is a relatively weak acid and at physiological pH only the first acidic dissociation equilibrium is significantly populated. Production of CO2 implies acido-sis of the biological medium; however, carbon dioxide is removed by diffusive processes across the respiratory epithelia like the O2 uptake. Thus, the respiratory system represents the main, but not exclusive, excretory route for CO2 removal. The pH homeostasis is also achieved by H+ and HCO3- regulation by the kidneys as described below. The solubility of CO2 in body fluids is increased by its hydration reaction to form carbonic acid. The enzyme carbonic anhydrase plays an important role in catalyzing the conversion reaction of CO2 to carbonic acid and the back reaction of the chemical equilibrium. Bicarbonate represents the main form of carbon dioxide at the average pH of body fluids (7.4). Carbonic anhydrase is abundant in erythrocytes, in the epithelial cells of the respiratory surfaces, and the nephron.

Different molecules represent the terminal products of nitrogen-containing groups. The primary product of deamination reactions is ammonia. This term includes both the nonionic ammonia (NH3) and ammonium ion (NH|), respectively. In the environment, ammonia undergoes a six-electron oxidation to nitrite and then a two-electron oxidation to nitrate by nitrifying bacteria. Other widely represented compounds involved in the nitrogen waste are urea and uric acid while in some taxa, allantoin, trimethylamine oxide (TMAO), guanine, creatinine, hippuric acid, and aminoacids are involved (Table 1).

The animals that excrete ammonia, urea, or uric acid are termed ammoniotelic, ureotelic, and uricotelic, respectively. The selection of a given product as nitrogen excretory molecule results from a tradeoff between the toxicity of the compound (hence the need to dilute it in body fluids to tolerate its presence before excretion) and the complexity of the molecule and of the metabolic pathway to synthesize it (hence the energetic investment by the organism). In the context of toxicity, water availability represents an important environmental selective factor. Ammonia is toxic for animals due to its strong alkaline reaction (pKa 9.5); furthermore, the hydrated NH| ion has the same ionic radius as hydrated K+; thus, NH| can affect the membrane potential of cells and, in particular, of axons. Ammonia can affect a number of metabolic and physiological processes, apart from its effect on pH: it affects the brain-blood barrier; in astrocytes, it induces accumulation of glutamine and affects phagocytosis and endocytosis; it may mediate excessive activation of glutamatergic signaling. Its critical concentration level for mammals is around 0.05 mM and to keep its concentration low, animals have to use about 0.51 of water to excrete 1 g of nitrogen in this form. Urea requires 10 times less water per gram of nitrogen than ammonia, whereas insoluble uric acid and guanine require 50 times less water. As a result, aquatic animals, including invertebrates and terrestrial animals that live in humid environments, excrete mostly ammonia. Terrestrial animals that live in arid environments, where water conservation is a severe problem, excrete urea, uric acid, or guanine. The ontogenetic traits of nitrogen excretion of the frog Rana catesbeiana provide an interesting example on the correlation between nitrogen end products and environmental water availability: as a tadpole this vertebrate lives in water and excretes the nitrogen wastes as ammonia, whereas during the metamorphosis it expresses the enzymes responsible for urea synthesis and the product for nitrogen waste becomes urea. The pathways of nitrogen excretion may vary also at the level of individuals as a function of water availability in the case of vertebrates inhabiting environments where water availability may change drastically with season. A typical example is given by the African lungfish that are ammoniotelic during the immersed lifetime. Individuals undergoing estivation in dried ponds become ureotelic and safely tolerate high concentrations of urea in their body fluids for the whole period of scarce water. With the return of rain, the body absorbs water that is used to excrete excess urea. Similar cases are offered by amphibians and reptiles that excrete ammonia in acquatic habitat or urea or uric acid on land.

As the complexity of the molecule increases, excretion requires an energy investment with respect to both the synthetic pathways and the energy of chemical bonds that is lost when the molecule is excreted. For a quantification of such energetic balance, in Table 2 the heat of combustion -AH (that reports on the energy still present in the chemical bonds of the excreted compound) and the Gibb's free energy of formation -AG (that reports on the energy invested in the synthesis) are reported for the main excreted molecules.

As evident, in going from ammonia to uric acid, the organism has to invest more energy for the synthesis of the excreted compound. Furthermore, the cost of nitrogen excretion increases in the same direction also as far as the energy content of the excreted molecule and the concomitant waste of carbon atoms are concerned. Yet, the toxicity of the molecules decreases. The strategies involving ammoniotelism, ureotelism, and uricotelism, therefore, can be interpreted as a tradeoff between energy investment and toxicity of the synthesized compound (hence water investment).

Ammonia is formed during amino acid catabolism, usually in the liver. The a-amino group is transferred to a-ketoglutarate by transaminase enzymes to form L-glutamate. This product is moved from cytosol to mitochondria where it is oxidatively deaminated by L-glutamate dehydrogenase (GDH) to form ammonium ion and a-ketoglutarate. In mammals this enzyme can use either NAD or NADP+. The a-ketoglutarate can be used in the citric acid cycle (tricarboxylic acid cycle (TCA)) for glucose synthesis; therefore, GDH is an ubiquitous enzyme in both higher and lower organisms. The overall ammonia formation reaction is given in Scheme 1.

Many animals also produce ammonia in the kidney by glutaminase during the degradation of glutamine. Ammonia produced in the muscle is added to pyruvate to form alanine in the glucose-alanine cycle. Alanine is taken up by the liver where it undergoes oxidative dea-mination described above. Ammonia can also be produced from urea (the synthesis of this product is described below) by urease present in some mollusk and coral tissues, in the liver of two Carcharhinid sharks, in bacteria, yeast, higher plants, and in some mammals. During this process urea is hydrolyzed to carbon dioxide and ammonia.

Urea is synthesized in the liver via ornithine-urea cycle (Scheme 2).

In this cycle two amino groups and one carbon dioxide molecule are added to an ornithine molecule to form arginine. This is converted to ornithine and urea in the presence of the enzyme arginase. The conversion of argininosuccinate into arginine also produces fumarate

Table 1 Main nitrogen-containing compounds excreted by invertebrates and vertebrates

Ammonia Urea Uric acid Guanine Creatine Creatinine Allantoin Hippuric acid TMAO Amino acids

Skeletal Structure For Ch4n2o

CH4N2O (60.06)

C5H4N4O3 (168)

C5H5N5O (151.1261)

"OOCx /NHJ

"OOCx /NHJ

CH4N2O (60.06)

C5H4N4O3 (168)

C5H5N5O (151.1261)

C4H9N3O2 (131.13)

C4H7N3O (113.245)

C4H6N4O3 (158.1164)

C9H9NO3 (179.17)

The molecular weights in g mol 1 are assumed in parentheses. For amino acids an average molecular weight of 110 g mol 1 is given.

Table 2 Properties of the main nitrogen-containing excreted waste products

(water investment)

Molecular formula

(MW)

C/N ratio

-AH (kcal mol-1)

(kcal mol—l)N-tom

(kJmor^N-U

Ammonia

52.4

High

NH3

0

90

90

6.3

6.3

(high)

(17-18)

Urea

39.8

Moderate

(NH2)2CO

0.5

152

76

48.7

24.4

(moderate)

(60)

Uric acid

0.0015

Low

C5H4N4O3

1.25

460

115

91.46

22.87

(low)

(168)

aHeat of combustion. bGibb's free energy of formation.

aHeat of combustion. bGibb's free energy of formation.

COO-C-O

COO-a-Ketoglutarate

COO-

L-Amino acid

Transaminase

COO-

COO-L-Glutamate

a -Keto acid

+NH3

COO-

COO-L-Glutamate

COO-CO CH2 CH2

COO-L-Ketoglutarate

Scheme 1 General pathway for amino acid transamination and oxidative deamination responsible for the formation of free ammonia, during amino acid metabolism. The deamination reaction of glutamine, the glucose-alanine cycle, and the hydrolysis of urea by urease, also responsible for ammonia productions, are not included.

that is a key element in the TCA. Two adenosine 5'-triphosphate (ATP) molecules are used in the first step of the cycle in the formation of the carbamoyl phosphate and arginiosuccinate. Therefore, the formation of urea is an irreversible process. The overall energetic costs are reduced by the production of NADH by the hydration of fumarate to L-malate in TCA.

An important source of ornithine sustaining the urea cycle is the reaction pathway that produces creatine, synthesized mainly in the liver from arginine, glycine, and methionine by two enzymes: glycine amidi-notransferase and guanidinoacetate N-methyltransferase. About 95% of creatine and its phosphorylated form, phosphocreatine, is stored in skeletal muscles (Scheme 3).

Urea can be produced also via uricolysis of uric acid by urate oxidase to form allantoin. Humans, birds, and reptiles do not have this enzyme and the reaction is stopped at uric acid, whereas other mammals are able to produce allantoin which can be further decomposed to form urea (Scheme 4). The toxicity of urea has been discussed above; however, it is worth noting that in some animals, notably elasmobranches, the concomitant presence of TMAO (at a ureaTMAO ratio of 2:1) counteracts such toxic effects and urea concentration can be as high as 0.05-1 mM. Also xanthine and hypoxanthine are oxidized by xanthine oxidase enzyme to form uric acid. These reactions are involved in the purine degradation process and the catabolic pathway of purine nitrogen converges to

Citrulline> HC—COO-

Citrulline> HC—COO-

Arginine Uric Acid Cycle

Argininosuccinate | +

HC-COO-

i Arginino-succinate synthetase

Aspartate

Cytosol

Arginino-succ liase

Arginine

Arginase

Arginino-succ liase

Citric acid cycle

H2N C nh2 Urea

Scheme 2 Reaction pathway responsible for the urea synthesis in liver (ornithine-urea cycle). This cycle receives the input of ornithine from the creatine synthetic pathway described in Scheme 3 and provides the fumarate substrate for the citric acid cycle.

Urea cycle

Arginine

COO-

COO-Glycine

Ornithine NH2 C = NH

Glycine amidinotransferase

Guanidinoacetate N-methyltransferase

Creatine kinase

N-CH

COO-Phosphocreatine

Scheme 3 Creatine and phosphocreatine synthetic pathway producing ornithine as side product. The latter molecule sustains the ornithine-urea cycle described in Scheme 2.

COO-Guanidinoacetate

COO-Creatine that of the amino acid nitrogen. In many human tissues, Hippuric acid is a nitrogen compound found in the uric acid is a powerful radical scavenger and antioxidant. urine ofhorses and other herbivores. It is produced by the The xanthine oxidase enzyme is missing arachnids and so reaction of benzoic acid or toluene with glycine. A deri-

their major excretory product is guanine. vative of this acid is the ^-amino-hippuric acid (PAH).

C NH

HC COO

NH3+

C NH

Synthesis Uric Acid

Scheme 4 Production of uric acid through the purine degradation, oxidative pathway. The pathway included in the red box shows the further conversion of uric acid to yield other catabolites important for nitrogen excretion, allantoin and urea.

Another metabolite of benzoic acid is the ornithuric acid; it is found in the excretion of chicken, gecko, and side-necked turtle.

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