Introduction

Ammonia production occurs in all tissues of the body during the metabolism of a variety of compounds. Ammonia is produced by metabolizing amino acids and other compounds containing nitrogen. Ammonia exists as an ammonium ion (NH4⁺) at the physiological pH. It is produced in our body mainly by transamination followed by deamination from biogenic amines, from amino groups of nitrogenous bases like purine and pyrimidine, and in the intestine by intestinal bacterial flora through urease action on urea. Ammonia disposal takes place primarily by the hepatic formation of urea. The blood level of ammonia must remain very low because even slightly elevated concentrations (hyperammonemia) are toxic to the central nervous system. A metabolic mechanism exists by which nitrogen is moved from peripheral tissues to the liver for its ultimate disposal as urea while maintaining low circulating ammonia levels.

Fundamentals

The amino acids participate in certain common reactions like transamination and deamination to 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 to liberate free ammonia to synthesize urea. 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, which 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. It occurs in nearly all tissues of the body. Ammonia is unloaded via glutaminase by a reaction, glutamine --> NH_³ + glutamate. It specifically occurs in the kidneys and intestine and in very low concentrations in the liver. This reaction is induced by acidosis.

Cellular Level

In nature, ammonia exists as both NH³ and in the ionic form of ammonia as ammonium ion (NH⁴⁺). A buffering reaction: NH³⁺ H⁺ --> NH⁴⁺ is used to maintain the relative amount of each form. Under biological conditions, the pKa of this reaction is about 9.15, and this reaction occurs almost instantaneously. As a result, most ammonia under physiological conditions exists as NH^4+, and only about 1.7% of total ammonia presents as NH³ at pH 7.4. Ammonia is a very small, uncharged particle. Due to this character of ammonia, it was initially believed that ammonia is highly permeable across the lipid membrane because of the maintenance of proper diffusion equilibrium. However, after thorough studies, this was refuted. Instead, it was seen that though ammonia is an uncharged particle, the asymmetrical arrangement of positively charged hydrogen ions around a central nitrogen molecule converts this ammonia molecule into a relatively polar particle. Ammonia has a molecular dipole moment of 1.47 D, which indicates the degree of separation between positively and negatively charged particles. 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 typical characteristic of permeability results in developing a transepithelial gradient of ammonia, which is demonstrated to be present in the kidneys. Without specific transport proteins, ammonia also has a restricted permeability property across lipid bilayers. Due to the inability to transport ammonia through the lipid bilayer in the plasma membrane, the hypothesis of transporting NH4+ by “NH4+ trapping” was introduced. However, the accuracy of this concept has not been fully established. Ammonium ion (NH⁴⁺) has poor permeability across the biological membrane without an appropriate transporter. Some tissues have no detectable permeability, such as the apical membrane used to collect duct segments. However, the transport of ammonium ions (NH4+) across the biological membrane can occur by specific proteins and is particularly crucial for renal ammonia excretion. Due to the particular biological character of ammonium ions in hydrated form, these proteins can be used to transport this specific ion. When examined in aqueous solutions, the ammonium ion (NH⁴⁺) and potassium ion (K⁺) show almost identical biophysical characteristics. This particular unique character allows ammonium ions to be effectively transported at the transport site of potassium ions.[1]

Function

In response to an acid challenge, the production of ammonia and its excretion are major mechanisms by which the kidney produces bicarbonate.[2]Under physiological conditions, when the body is exposed to an acidic environment, the kidney stimulates the production of ammonia and its excretion. 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 in this site depends not only on the sufficient delivery of glutamine but also on the ability of the proximal tubule to take up that particular glutamine delivered. The acidotic condition stimulates the delivery and augments glutamine transport into the kidney. SNAT3/Slc38a3 is a glutamate transport protein, and the amount of this increases with an increase in glutamine uptake, resulting in acidosis. Enzymes responsible for ammonia production are upregulated by the acidotic condition that leads to augmented ammonia production from proximal tubules of the kidney. This acidosis also stimulates increased ammonia secretion into the lumen, resulting in increased transport of ammonia towards the thick ascending limb, leading to enhanced absorption and formation of ammonia in the medullary interstitium.[3]

Testing

It is clinically relevant to determine the level of ammonium in the urine to determine the capacity of kidneys for an appropriate response 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 the estimation of ammonia excretion through the kidney. 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 beneficial based on the assumption that urinary ammonium ion is excreted only in association with the chloride ion. However, this method is not useful for other ions like sodium, potassium, glucose, and urea nitrogen. For this, urinary osmolal gap estimation 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 like mannitol or unmeasured cations, the urinary osmolal gap only shows the ammonium ion concentration with its anion. However, the gold standard of measurement of urinary ammonium ion is the same as the enzymatic assay to measure the blood ammonium ion levels.[4]

Clinical Significance

In chronic kidney diseases (CKD), the kidney cannot produce and excrete an adequate quantity of ammonia, leading to acid retention and metabolic acidosis.[5] With the progression of kidney disease, the glomerular filtration rate simultaneously falls, 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 the formation of ammonia in normal individuals but not in patients with CKD, although the serum level of glutamine is high in both cases. This unique phenomenon in the case of CKD exists due to the reduction of glutamine transporter SNAT3/Slc38a3.[4] Studies performed in nephrectomized rats show that other defects can be seen in the production of ammonia and transport. Researchers found in the animal model of CKD that despite the urinary acidification, the defect was in the net excretion amount of acid. In comparison to normal control, it was also seen that the delivery of ammonia shows a marked elevation at the peripheral accessible portion of the proximal renal tubule. Research also observed that ammonia is at a lower concentration in the loop of Henle, which allows for the escape of more ammonia mainly from the cortex of the nephron, and it then enters back in the renal vein and returns to the central circulation. This property decreases the amount of ammonia in the medullary interstitium, leading to a decreased concentration gradient between the medullary 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 that leads to increased reabsorption of bicarbonate, reduction of formation of titratable acid, and secretion of ammonia. Recent studies in the polycystic kidney model show that the decrease in ammonia excretion in urine is due to the decrease of ammonia transporter called RhCG. However, this hypothesis has been refuted with the findings achieved in the remnant kidney, which shows that the distribution of RhCG transporter protein increases in the apical and basolateral portions. So, in patients with chronic kidney disease, despite the presence of acidosis, the production and excretion of ammonia are seen to be reduced. Thus, normal acid-base balance is disrupted in the case of chronic kidney diseases.[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. Though it is unclear what the exact mechanism of myopathy is, the assumption is that ammonia detoxification takes precedence over protein synthesis in muscles. Elevated ammonia levels have also been proposed to increase muscle breakdown through the activation of 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 the decreased capacity of detoxication results due to dysoxia of these cells.[8]