Disturbances of the urea cycle and ammonia detoxification


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      Proteins ingested with the diet are digested to aminoacids by endopeptidases (e.g. pepsin, trypsin and chymotrypsin) and by exopeptidases (carboxypeptidases and aminopeptidases) and absorbed by the small intestine. They constitute the major source of nitrogen in our metabolism. Endogenous proteins are degraded to aminoacids by several systems (e.g. the proteasome, and the lysosomes); these may be used to produce new proteins or can be directed to the Krebs cycle to produce energy.
      Since the Krebs cycle does not consume nitrogen, the catabolism of aminoacids requires the removal of the amino group. This is achieved by enzymes called transaminases (or aminotransferases). There are as many transaminases as there are aminoacids, and each uses α-ketoglutaric acid (a metabolyte of the Krebs cycle) as the acceptor of the amino group; the reaction of Alanine Aminotransferase (ALT; also called Glutamic Piruvic Transaminase, GPT) is as follows:

      The reaction product is piruvate, which proceeds to the Krebs cycle after decarboxylation by piruvate decarboxylase. The reactions catalyzed by transaminases tend to accumulate glutamic acid and to subtract α-ketoglutarate from the Krebs cycle. Recovery of α-ketoglutarate is achieved by oxidative deamination. The reaction, catalyzed by glutamate dehydrogenase, releases free ammonia:

      α-aminoacids are thus converted to the corresponding α-ketoacids, and their amino group is incorporated into glutamate. As we shall describe in detail below, in all mammals half the glutamate is processed by glutamate dehydrogenase and its nitrogen released as ammonia; the other half is used as a donor of ammonia to oxalacetate, which is converted to aspartate.
      Some animals (notably fishes) eliminate ammonia in the urine: they are defined ammoniotelic (or ammonotelic). Unfortunately, ammonia is basic and at high concentration it is toxic for the renal tubules: thus animals not living in water must convert this compound to something less toxic in order to be able to concentrate it in the urine a to save water. Mammals are defined ureotelic because they use urea as the terminal metabolyte of nitrogen, and their blood and urine contain this compound as the main source of non-protein nitrogen. The transformation of ammonia into urea is carried out by the liver, via a metabolyc pathway called the urea cycle:

      The first reaction (catalyzed by the ezyme carbamoyl phosphate synthetase) uses bicarbonate, ammonia and 2 molecules of ATP to produce the high energy intermediate carbamoyl phosphate. Carbamoyl phosphate is then combined with a molecule of the non-proteic aminoacid ornithine to produce the non-proteic aminoacid citrulline; the enzyme is called Ornithine transcarbamoylase. Citrulline is combined with a molecule of aspartic acid by the enzyme Arginino-succnate synthetase; the product is argininosuccinic acid. The enzyme Arginino-succinate lyase breaks down this molecule to yield arginine (a proteic aminoacid) and fumarate (a Krebs cycle metabolyte). Arginase, the last enzyme of the cycle hydrolyzes a C-N bond of arginine to produce urea and ornithine, than can be used in a new cycle.
      The overall balance of the urea cycle (neglecting the consumption of 3 ATP) is as follows:

      An interesting observation is that only one of the nitrogens of urea comes from ammonia (via glutamate dehydrogenase); the other derives from one molecule of aspartic acid. Clearly aspartic acid is not enough to sustain the urea cycle: it accounts for only some 7% of the total aminoacids of alimentary proteins. Where does aspartic acid come from? The amino groups of all aminoacids are transferred to α-ketoglutarate by the corresponding aminotransferase, and aspartate is no exception. However the transaminase reactions are all reversible and in the case of Aspartate Aminotransferase (AST; also called Glutamate Oxaloacetate Transaminase, GOT) the reaction flows mainly backwards:

      As a consequence, aspartate is produced from glutamate and oxaloacetate (a Krebs cycle metabolic intermediate) and the two nitrogens of the molecule of urea derive from all aminoacids that our organism catabolyzes, via transamination to glutamate and aspartate, as schematically depicted in the figure below that shows the flow of nitrogen from aminoacids to urea (in red the key metabolytes).

Audio: Metabolism of ammonia

      The α-ketoacids obtained from transamination are further metabolized and are converted to acetyl-CoA or to intermediates of the glycolysis or the Krebs cycle. The relationships between these catabolic pathways is fascinating (but quite intricated):


      Nitrogen is contained in the blood in two major forms: urea and proteins. Less relevant sources are free ammonia, free aminoacids, hormones and creatinine. The normal range for BUN is 6-20 mg/dL. It is important to remark that, given the chemical formula of urea (H4CN2O, MW=60) and its nitrogen content (47% w/w), 6-20 mg/dL of BUN correspond to 13-43 mg/dL urea.
      The easiest method to determine BUN is as follows: (i) serum is obtained from venous puncture; it may be de-proteinized using TCA precipitation followed by neutralization. (ii) The concentration of free ammonia is selectively measured using the appropriate electrode. (iii) The bacterial enzime urease is added to the sample; it catalyzes the decomposition of urea to carbon dioxide and ammonia: CO(NH2)2 + H2O --> CO2 + 2 N3. (iv) The concentration of ammonia is again determined with the same electrode; BUN corresponds to the increase of ammonia concentration after addition of urease.
      In alternative several colorimetric methods are available, based on the Berthelot's reaction: urea is degraded to carbon dioxide and ammonia by urease and ammonia is allowed to react with hypochlorite and phenol (in the presence of a catalyst) to yield the blue compound indophenol, whose concentration can be determined by absorbance spectrophotometry.

      Creatine and creatinine. Creatine is synthesized by the liver starting from arginine, glycine and S-adenosilmethinine. The daily production of creatine is approximately 1 g/die. This compound is used mainly by the skeletal muscle as a reservoir of high energy phosphate bond, in the form of phosphocreatine (or creatine phosphate), that can rapidly and anaerobically replenish ATP (for burst energy consumption). Phosphocreatine may undergo spontaneous conversion to the terminal metabolyte creatinine, which cannot be utilized by the muscle and is secreted in the blood and eliminated in the urine. The formulas and reactions are as follows:

      Creatinine is the second most important low-molecular weight nitrogen containing compound in the blood. In the healthy adult its serum concentration is 0.5-1.2 mg/dL (or 50-110 μMol/L).

Reference values for low molecular weigth nitrogen-containing compounds in human plasma
urea < 20 mg/dL by nitrogen (< 43 mg/dL for urea molecule; < 7 mMol/L)
creatinine < 1.2 mg/dL (< 110 μMol/L)
ammonia < 50 μMol/L (in newborns and infants < 150-200 μMol/L)

      It is perhaps surprising that a metabolism so fundamental as the urea cycle is so prone to severe genetic defects, given that these diseases usually cause early death and are strongly counter selected. However one should consider that (i) insufficiency of dissimilatory pathways (such as the urea cycle) is usually silent in the foetus, as the mother body takes care of the excretion of the accumulated metabolyte; and (ii) these diseases are recessive and in some cases X-linked; thus the affected genes are to some extent protected from severe selection.
      As a general rule, ereditary defects of the urea cycle become evident in the newborn, and have a very severe prognosis, survival beyond the infantile age being uncommon. They are inherited as autosomal recessive traits, with the exception of the deficiency of ornithine transcarbamoylase that is recessive and X-linked. The symptoms of these defects are essentially the same: no matter which enzyme is involved, hyperammoniemia is constantly present and is the leading cause of the clinical symptoms: mental retardation, coma, and death. Seizures and vomiting are often observed. The normal value of ammoniemia in the adult's blood is < 50 μMol/L; in the newborn it may be up to 150-200 μMol/L because of the incomplete maturity of the liver. In addition to hyperammoniemia, genetic defects of the urea cycle may cause some specific aminoacid to appear in the blood and urine at abnormally high concentrations (e.g. citrullinemia, argininosuccinic acidemia, argininemia). Diagnosis is suspected on the basis of clinical findings and demonstration of hyperammoniemia, and confirmed by gene sequencing.

Defects of the urea cycle
Disease name Enzymatic defect Organs affected and symptoms
Deficiency of Carbamoyl phosphate synthetase   Hyperammoniemia, vomiting, mental retardation, coma and death
Deficiency of N-acetyl glytamate synthase (N-acetyl glutamate is a required allosteric activator of carbamoyl phosphate synthase)   Simlar to the deficiency of Carbamoyl phosphate synthetase
Deficiency of Ornithine transcarbamoylase   Hyperammoniemia, vomiting, mental retardation, coma and death (X-linked; lethal in the male)
Citrullinemia Deficiency of Argininosuccinate synthetase Hyperammoniemia, vomiting, mental retardation, coma and death
Type II citrullinemia (adult onset) Deficiency of the Aspartate/Glutamate carrier 2 (AGC2, citrin), a mitochondrial transporterSimilar to, but less severe than, citrullinemia. Patients usually reach adulthood. Crises of hyperammonemia may be precipitated by carbohydrate intake
Argininosuccinic acidemia Deficiency of Argininosuccinate lyase Hyperammoniemia, vomiting, mental retardation, coma and death
Argininemia Deficiency of Argininase Hyperammoniemia, convulsions, spasticity, mental retardation, coma and death
Deficiency of ornithine transaminase   Hyperammoniemia of variable severity, convulsions, mental defects

Audio: Hereditary defects of the urea cycle

      The urea cycle uses up to 80 or 90% of the ammonia derived from aminoacid metabolism, either as ammonia or aspartate. The remaining fraction of ammonia is utilized by other metabolic pathways, that produce aminoacids. Examples are the biosynthesis of Glycine by Gly synthase and that of Glutamine by Gln synthase.
Because of these additional pathways, genetic defects of the urea cycle are not the only possible cause of hyperammonemia, and milder, but still symptomatic, levels of hyperammonemia may be observed in several genetic defects of aminoacid byosynthesis. Moreover, glutamine, like aspartate, is a common donor of ammonia for the biosynthesis of nucleotides: for example the conversion of UTP to CTP utilized Gln as the donor of amine group. As a consequence, the (rare) genetic defect of Gln synthase may cause the accumulation of nucleotide base precursors whose further conversion is prevented by lack of Gln.
      The differential diagnosis of hyperammonemias relies on the identification of other metabolytes that may be present in excess or in defect; these may be intermediates of the urea cycle (e.g. argininosuccinic acid), or intermediates of other pathways.
Differential diagnosis of hyperammonemias
In the adultIn the newborn
Acute or chronic liver failure
(due to viral hepatitis, cirrhosis, intoxication, etc.)
Sepsis, enteritis
(NH3 produced by bacteria; inactivation of Gln synthase)
Inherited defects of the ammonia cycle
Inherited defects of Gln synthase
Inherited defects of organic acid metabolism
Liver prematurity

      Some important associations may be noticed: hyperammonemia may be associated to abnormal levels of nucleotide biosynthesis intermediates (e.g. orotic acid) or intermediates of the aminoacid catabolism. These associations are usually mediated by the leakage to the cytoplasm of carbamoyl phosphate accumulated in the mitochondrion because of the blockade of the urea cycle. Remember that under physiological conditions the carbamoyl phosphate required for pyridine nucleotide biosynthesis is produced by the cytoplasmic synthase, that for the urea cycle by the mitochondrial synthase; only under pathological conditions does mitochondrial carbamoyl phosphate leak to the cytoplasm. When this occurs, the biosynthesis of pyrimidine nucleotides is stimulated, leading to an atypical form of orotic aciduria (to be distinguished from the usual form, due to a genetic defect of orotate PRPP transferase), in which hyperammonemia and excess orotic acid are both present. Defects of the metabolism of aminoacids and other organic acids may cause hyperammonemia by interfering with the mitochondrial transport or by inhibiting enzymes relevant to the urea cycle (e.g. propionyl-CoA inhibits carbamoyl phosphate synthase I). A list of the metabolic defects that may be associated to hyperammonemia includes: propionic acidemia (defect of propionyl-CoA carboxylase) and methylmalonic acidemia (defect of methylmalonyl-CoA mutase); isovaleric acidemia; and glutaric acidemia. In all these cases, a hemogas analysis is indicated because it may reveal the coexistence of hyperammonemia and metabolic acidosis with increased anion gap. The differential diagnosis is important because some of these defects may respond to dietary and vitamin treatment.

      Urea is produced by the liver (for over 90%; other organs, notably the kidney produce less than 10%) and excreted in the urine by the kidney.

     Advanced states of liver failure may cause reduced production of urea and very low BUN. This condition is only observed when the liver function is compromised to a great extent, because the ability of the liver to produce urea greatly exceeds the normal body requirements. Thus ther symptoms of liver failure will be present and will preceed the reduced biosynthesis of urea: hyperbilirubinemia with jaundice, reduced production of serum proteins (hypoalbuminemia), etc. Reduced production of urea is accompanied by hyperammoniemia, with the symptoms and laboratory signs described above. The other possible causes of reduced urea production are the inherited enzymatic diseases listed above. These are diagnosed shortly after birth, thus are neonatal diagnoses, not to be considered in adult patients. Hyperammonemia and encefalopathy are invariably present.

      Increased BUN usually points to kidney failure, and indicates measurement of renal function. Since urea and creatinine are cleared by the kidney, an increase of the serum concentration of these substances is usually indicative of kideny disfunction. Azotemia is essentially a synonimous of BUN, and hyperazotemia is the clinical condition in which azotemia is increased above normal values.
      Creatinine clearance. The kidney's ability to excrete creatinine is easily measured and provides an important estimate of the organ's function. The method takes advantage of the almost constant concentration of creatinine in the serum, due to the constant rates of production and excretion. A sample of urine is collected over a precisely measured time (e.g. one hour), preferably by a catheter. The concentration of creatinine is measured in the serum and in the urine. Given that the volume of urine can be precisely determined, one can calculate the moles or milligrams of creatinine excreted in one minute by the kidneys:
moles of creatinine excreted / minute = [urinary creatinine] x volume of urine / time
The amount of creatinine thus calculated was contained in the volume of serum filtered by the kidneys in one minute, i.e.:
volume of serum filtered / minute = moles of creatinine excreted in one minute / [serum creatinine]

      The normal clearance of creatinine is 90-120 mL / minute; since creatinine is neither reabsorbed nor excreted by the renal tubuli, this value equals the volume of serum filtered by the glomeruli in one minute (Glomerular Filtration Rate, GFR). The GFR decreases physiologically with age, and in patients above 70 it is not uncommon to find GFR values of 60 mL/min. Renal atrophy, polycystic kidney disease, glomerulonephritis, aterosclerotic kidney disease are common causes of reduced glomerular filtration. It is important to remark that the volume of urine produced in one minute is highly variable and does not provide any information on glomerular filtration: the kidney can reabsorb up to 99% of the filtered water, depending on the amount of water the patient drinks and the osmotic effect of urine solutes.

Further readings
Savy et al. Acute pediatric hyperammonemia: current diagnosis and management strategies. Hepat Med. 2018; 10: 105–115.
J Häberle, A Chakrapani, N Ah Mew, and N Longo Hyperammonaemia in classic organic acidaemias: a review of the literature and two case histories
DB Flannery, YE Hsia, B Wolf Current status of hyperammonemic syndromes. Hepatology 1982; 2: 495-506.
SC Sreenath Nagamani, A Erez, and B Lee Argininosuccinate Lyase Deficiency.

Questions and exercises:
1) Hyperammoniemia suggests:
An hereditary defect in aminoacid metabolism
An hereditary defect of the urea cycle
An hereditary defect of aminoacid transport

2) Phenylketonuria is among the most common hereditary defects of metabolism; the aminoacid whose metabolism is impaired is/are:
Phenylalanine and tyrosine

3) Inherited defects of aminoacid metabolism may cause metabolic acidosis. In these cases the anion gap is

4) All genetic defects of the urea cycle are inherited as autosomal recessive traits, except for the following one, which is sex-linked:
deficiency of ornythine transcarbamoylase
arginino succinic acidemia

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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).

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