Biochemistry, Ammonia

Fundamentals

The amino acids participate in common reactions such as transamination and deamination, which produce ammonia. The amino group of amino acids is utilized to form urea, an excretory product for protein metabolism. The amino acid is transaminated to produce a molecule of glutamate. Glutamate is the amino acid that undergoes oxidative deamination, liberating free ammonia for urea synthesis.

Once free ammonia is formed in peripheral tissues, it must be transferred to the liver for conversion to urea. This is carried out by the “glucose-alanine cycle.” In the glucose-alanine cycle, alanine, formed by the transamination of pyruvate, gets transported in the blood to the liver, where it is transaminated by alanine transaminase to pyruvate. The non-toxic storage and transport form of ammonia in the liver is glutamine.

Ammonia is loaded via glutamine synthetase by the NH³ + glutamate → glutamine reaction. This reaction occurs in nearly all tissues of the body. Ammonia is unloaded via glutaminase by the reaction glutamine --> NH3 + glutamate. This specifically occurs in the kidneys and intestine, and at very low concentrations in the liver; it is induced by acidosis.

Cellular Level

In nature, ammonia exists as both NH³ and the ionic ammonium ion (NH⁴⁺). A buffering reaction, NH3 + H+ --> NH4+, is used to maintain the relative amounts of each form. Under biological conditions, the pKa of this reaction is approximately 9.15, and it occurs almost instantaneously. As a result, under physiological conditions, most ammonia exists as NH4+, and only about 1.7% of the total ammonia is present as NH³ at pH 7.4.

Ammonia is a very small, uncharged particle. Due to this characteristic of ammonia, it was initially believed to be highly permeable across lipid membranes because it maintains proper diffusion equilibrium. However, after thorough studies, this was refuted. Instead, it was observed that although ammonia is an uncharged particle, the asymmetrical arrangement of positively charged hydrogen ions around the central nitrogen atom makes it relatively polar.

Ammonia has a molecular dipole moment of 1.47 D, indicating the degree of separation between positively and negatively charged regions. In contrast, HCl has a dipole moment of 1.08, and the water molecule has a dipole moment of about 1.85. Due to this charged polarity, ammonia has limited and minimal permeability through lipid membranes.

This characteristic permeability results in the development of a transepithelial ammonia gradient, as demonstrated in the kidneys. Without specific transport proteins, ammonia also has limited permeability across lipid bilayers. Because ammonia cannot cross the lipid bilayer of the plasma membrane, the hypothesis of NH4+ transport via “NH4+ trapping” was proposed. 

The accuracy of this concept has not been fully established. The ammonium ion (NH4+) has poor permeability across biological membranes in the absence of an appropriate transporter. Some tissues have no detectable permeability, such as the apical membrane used in collecting duct segments.

However, the transport of ammonium ions (NH4+) across biological membranes can occur via specific proteins and is particularly crucial for renal ammonia excretion. Due to the distinctive biological properties of hydrated ammonium ions, these proteins can be used to transport this ion. When examined in aqueous solutions, the ammonium ion (NH⁴⁺) and the potassium ion (K+) exhibit nearly identical biophysical properties. This unique character allows ammonium ions to be effectively transported at the site of potassium ion transport.[1]

Function

In response to an acid challenge, the production and excretion of ammonia are major mechanisms by which the kidney produces bicarbonate.[2] Under physiological conditions, when the body is exposed to an acidic environment, the kidneys stimulate the production and excretion of ammonia. The primary source of ammonia is glutamine, which is excreted in the urine.

The proximal tubule is the main site of ammonia formation, and the effective rate of glutamine delivery at this site depends not only on sufficient glutamine delivery but also on the proximal tubule's ability to take up the glutamine delivered. The acidotic condition stimulates the delivery and augments glutamine transport into the kidney. SNAT3/Slc38a3 is a glutamate transporter, and its abundance increases with increased glutamine uptake, thereby contributing to acidosis. Enzymes responsible for ammonia production are upregulated by acidotic conditions, leading to increased ammonia production in the kidney's proximal tubules. This acidosis also stimulates increased ammonia secretion into the lumen, thereby increasing ammonia transport towards the thick ascending limb, leading to enhanced absorption and formation of ammonia in the medullary interstitium.[3]

Testing

It is clinically relevant to measure urine ammonium levels to assess the kidneys' capacity to respond appropriately to an acid challenge. Kidneys excrete increased amounts of ammonia in acidotic conditions than in normal acid-base balance conditions. There are several methods for estimating ammonia excretion through the kidneys. One of the most appropriate and widely accepted methods is to measure the urinary anion gap and urinary osmolal gap. The urinary anion gap is determined as UNa⁺ + UK⁺ −UCl^-. This method is based on the assumption that urinary ammonium ions are excreted only in association with chloride ions. However, this method is not useful for other ions such as sodium and potassium, or for substances like glucose and urea nitrogen. For this, estimation of the urinary osmolal gap is necessary. 

The urinary osmolal gap is determined by Uosm−[2×(UNa⁺+UK⁺)+UUN/2.8+Uglucose/18)]Uosm−[2×(U+U)+U/2.8+Uglucose/18)]. One can assume that, in the absence of any osmotically active material such as mannitol or unmeasured cations, the urinary osmolal gap reflects only the ammonium ion concentration and its anion. However, the gold standard for measuring urinary ammonium ion is the same as the enzymatic assay to measure blood ammonium ion levels.[4]

Clinical Significance

In chronic kidney disease (CKD), the kidneys cannot produce and excrete an adequate amount of ammonia, leading to acid retention and metabolic acidosis.[5] As kidney disease progresses, the glomerular filtration rate declines, leading to increased production and excretion of ammonia by the remaining functioning nephrons. Subsequently, the remaining functioning nephrons cannot sustain the gradual increase of dietary acid load, leading to excessive acid retention inside the body.[6] 

In CKD, the kidney cannot take in or metabolize glutamine, the substrate for ammonia production. Glutamine uptake and metabolism contribute to only about 35% of ammonia production. The rest comes from other amino acids derived from the breakdown of peptide linkages. Further studies show that glutamine supplementation can increase ammonia formation in normal individuals but not in patients with CKD, although glutamine serum levels are high in both groups.

This unique phenomenon in CKD is due to reduced glutamine transporter SNAT3/Slc38a3.[4] Studies performed in nephrectomized rats show that other defects can be observed in ammonia production and transport. Researchers found in an animal model of CKD that, despite urinary acidification, the defect was in the net acid excretion. Compared with the normal control, the delivery of ammonia was also markedly elevated in the peripherally accessible portion of the proximal renal tubule.

Research also observed that ammonia is at a lower concentration in the loop of Henle, allowing more ammonia to escape mainly from the cortex of the nephron; it then enters the renal vein and returns to the central circulation. This property reduces the amount of ammonia in the medullary interstitium, thereby decreasing the concentration gradient between the interstitium and the collecting duct lumen. This specific defect of luminal entrapment of ammonia in the collecting duct is believed to correlate with distal delivery of bicarbonate, which leads to increased bicarbonate reabsorption, reduced formation of titratable acid, and increased ammonia secretion. 

Recent studies in the polycystic kidney model show that decreased ammonia excretion in urine is due to reduced expression of the ammonia transporter RhCG. However, this hypothesis has been refuted by the findings in the remnant kidney, which show that the distribution of RhCG transporter protein increases in the apical and basolateral portions. So, in patients with chronic kidney disease, despite acidosis, ammonia production and excretion are reduced. Thus, normal acid-base balance is disrupted in chronic kidney disease.[4]

Hyperammonemia (elevated ammonia concentration in systemic circulation above the normal range of approximately greater than or equal to 65 micromoles) correlates with liver failure and other significant causes of toxicity of skeletal muscle. So, liver disease associated with hyperammonemia is an apparent cause of muscle wasting disorders. A recent study showed that ammonia-lowering therapy in hyperammonemia portocaval anastomosis rat models improved the phenotype of muscle and metabolic activity of the protein.

Although the exact mechanism of myopathy is unclear, the assumption is that ammonia detoxification takes precedence over protein synthesis in muscle. Elevated ammonia levels have also been proposed to increase muscle breakdown by activating autophagy, contributing to the loss of muscle mass associated with cirrhosis. Additionally, alcohol correlates with an elevated level of serum ammonia, which can exacerbate the muscle protein metabolism impairment and elevate the risk of associated hepatic myopathy. This hypothesis supports the observation that patients suffering from alcoholic liver disease have a higher incidence and degree of muscle wasting than hepatic disease due to toxic or other infectious causes.[7]

Hemorrhagic shock is also known to be a cause of elevated blood ammonia levels. Excessive hemorrhage reduces the total hepatic blood flow, which causes ischemia in the periportal to the centrilobular area of the liver, and that leads to necrosis in patients in irreversible shock. The pericentral hepatocyte is responsible for glutamine synthesis, and the periportal hepatocyte is responsible for urea synthesis. High concentrations result from decreased detoxification capacity due to dysoxia in these cells.[8]