Hyperammonaemia in cats

The metabolism of ammonia is complex, yet a vitally important process required to maintain physiological homeostasis. Ammonia production is a by-product of the catabolism of nitrogen sources, most notably ingested protein and amino acids. Since ammonia is neurotoxic at high systemic blood concentrations, efficient mechanisms exist to compartmentalise the production and elimination of ammonia to maintain safe levels. The liver is the major player in ammonia excretion, while the intestine and kidney are the main ammonia producers. The liver can also generate ammonia, and the intestines and kidney can aid in ammonia excretion. Muscle can serve as an ammonia sink and in some cases increase ammonia load, which adds to the complexity of this process.

Ammonia metabolism and toxicity

Ammonia production

Ammonia is a biologically important source of nitrogen required for amino acid, protein and nucleic acid synthesis (Walker, 2014; Häberle, 2020; Butterworth, 2021). Additionally, ammonia plays a pivotal role in the kidney's maintenance of acid–base balance. Most ammonia is generated in the gastrointestinal tract from the action of urease-producing microbes on dietary and endogenous proteins (Table 1). The generated ammonia freely diffuses across the intestinal epithelium and makes its way into the portal blood. Additional sources of ammonia production include enterocyte metabolism of glutamine to glutamate in the intestine and kidney which releases ammonia, skeletal muscle production through the purine nucleotide cycle following strenuous exercise and hydrolysis of urea by urease-producing microbes in the gastrointestinal tract (Table 1).

Ammonia metabolism and elimination

Despite the critical role of ammonia in metabolism, high blood ammonia levels are cytotoxic (Auron and Brophy, 2012). The body rids itself of excess ammonia primarily by detoxification through the urea cycle in the liver and catabolism of glutamine in the liver, skeletal muscle and brain (Figure 1; Table 1). The urea cycle is a high-capacity system which converts ammonia into urea with the help of six enzymes and two transporters. In this cycle, ammonia and bicarbonate form carbamoyl phosphate via carbamoyl phosphate synthetase-1. This reaction requires N-acetylglutamate (acquired via a reaction catalysed by Nacetylglutamate synthase). Carbamoyl phosphate combines with ornithine in a reaction catalysed by ornithine transcarbamylase to form citrulline. Citrulline is transported to the cytosol and combines with aspartate to form argininosuccinate (this reaction is catalysed by argininosuccinate synthetase). Argininosuccinate is then cleaved by argininosuccinate lyase, yielding fumarate and arginine. Arginase cleaves arginine, producing urea and ornithine. The resultant urea is excreted in the urine, although some urea also diffuses back into tissues and fluids (Häberle, 2020; Lopes et al, 2022).

Ammonia that escapes metabolism in the urea cycle is handled by a low-capacity, but high-affinity, system that detoxifies it by incorporating ammonia as an amide group into glutamine, in a reaction catalysed by glutamine synthetase (Table 1). This buffering occurs primarily in the liver, brain and skeletal muscle. Glutamine is then released into the plasma. In this regard, glutamine serves to shuttle ammonia around in the body in a non-toxic form.

The major role of the kidney in ammonia metabolism is maintenance of acid–base balance; however, it only has a minor role in ammonia detoxification. The kidney can take up and deaminate glutamine to glutamate and the resultant ammonia can buffer free hydrogen by forming ammonium cation which is then excreted in urine. In this manner, the kidney can respond to metabolic acidosis and rid the body of ammonia at the same time (Walker, 2014).

Ammonia toxicity

When present in high concentrations, ammonia is neurotoxic. It can freely cross the blood–brain barrier and diffuse into the brain (Walker, 2014; Häberle, 2020). Once in the brain, ammonia is detoxified in the astrocyte by the conversion of glutamate to glutamine. However, accumulation of excess glutamine in the astrocyte increases intracellular osmolality and contributes to both low-grade cerebral oedema and cytotoxic damage. Ammonia neurotoxicity also involves alterations in neurotransmission through modulation of both inhibitory gamma-aminobutyric acid and excitatory glutaminergic neurotransmission. Acute ammonia toxicity affects glutaminergic receptors to a greater extent and thus clinical signs are more excitatory, while chronic ammonia toxicosis favours a more inhibitory phenotype, with activation of gamma-aminobutyric acid receptors and down-regulation of glutamate receptors. Since ammonia is also a potent inhibitor of the rate-limiting tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase, its toxicity also involves cytotoxic neural damage through impaired glucose oxidation and increased lactate production. Finally, hyperammonaemia activates microglial cells creating a pro-inflammatory environment. The morphological lesions associated with ammonia toxicity in the brain are varied but include cortical atrophy, basal ganglia lesions, neuronal loss, gliosis, focal cortical necrosis and myelination defects. Chronic hyperammonaemia gives rise to typical Alzheimer type II astrocyte lesions (Butterworth, 2021; Lopes et al, 2022).

Determination of blood ammonia concentration

Determination of blood ammonia is indicated in cats presenting with signs of a metabolic encephalopathy (Havig and Tobias, 2002; Zandvliet et al, 2005; Ruland et al, 2010; Sugimoto et al, 2018; Valiente et al, 2020). Commercial diagnostic assays for blood ammonia use straightforward enzymatic methods based on the glutamate dehydrogenase-catalysed reaction of the ammonium ion with α-ketoglutarate and nicotinamide or nicotinamide adenine dinucleotide phosphate. This reaction produces glutamate and the oxidised forms of nicotinamide and nicotinamide adenine dinucleotide phosphate. A corresponding decrease in absorbance at 340 nm is proportional to the blood ammonium concentration. Despite the ease of the actual assay, the measurement of blood ammonia is complicated by sample handling restrictions. Ammonia rapidly increases in whole blood and even in separated plasma with storage.

To protect against artifactually high ammonia values, blood must be drawn in a chilled ethylenediaminetetraacetic acid or heparin tube and immediately placed and kept on ice until spun down in a refrigerated centrifuge. Ideally, the plasma should be assayed immediately, but if this is not possible it should be frozen. Haemolysis must be avoided. These handling issues essentially restrict accurate blood ammonia determination largely to large institutions with adjoined laboratories that can perform in-house analysis. If shipped to a reference lab, the separated plasma should be frozen and shipped as soon as possible on dry ice, but even then, results may not be reliable.

Point of care blood ammonia analysers exist and allow rapid bedside determination in patients. The PocketChem BA (originally commercialised by the Menarini Group, Florence, Italy and now by Arkray, Inc, Kyoto, Japan) has been investigated for use in the dog and the cat (Goggs et al, 2008). This analyser relies on a colourimetric end point and uses only a small sample of whole blood. The analyser performed reasonably well when compared to reference assays, but at a cut-off of >100 umol/litre as abnormal, false negatives were common (13%) and false positives also occurred. The assay has a relatively narrow measurable range (7–286 um/litre) which limits its use in following response to therapy. Note also that there were only four cats in the test population in this study and the company has not published reference ranges for cats. As such, the value of blood ammonia determination with this bedside analyser is debatable.

Clinical presentation of hyperammonaemia in the cat

Clinical signs of hyperammonaemia in the cat are primarily related to neurotoxicity. Cats can present with lethargy, ataxia, altered levels of consciousness, seizures, cortical visual loss, failure to thrive and ptyalism. Collectively, these signs are referred to as hepatic encephalopathy. The signs are often episodic and, in some instances, can be precipitated by protein-loading (meals, gastroin-testinal bleeding), an anaesthetic event or an associated catabolic condition (infection, dehydration). Cats with congenital portosystemic shunting may also have copper-coloured eyes and will occasionally present with urethral obstruction because of ammonium biurate stones.

Causes of hyperammonaemia in the cat

A summary of reported causes of hyperammonaemia in the cat can be found in Table 2.

Liver bypass

Portosystemic bypass as a result of either congenital portosystemic shunting or multiple acquired portosystemic shunts leads to hyperammonaemia, as the portal blood containing large amounts of ammonia generated in the intestinal tract bypasses detoxification in the hepatic urea cycle. High blood ammonia concentrations are consistently reported in cats with congenital portosystemic shunting and multiple acquired portosystemic shunts (Havig and Tobias, 2002; Zandvliet et al 2005; Ruland et al, 2010; Tivers and Lipscomb, 2011; Sugimoto et al, 2018).

Evaluation of blood ammonia aids in the diagnosis of portosystemic shunting in the cat. The sensitivity and specificity of blood ammonia for the detection of congenital portosystemic shunting at a cut off of <50 umol/litre has been reported to be 83% and 76% respectively in cats (Tivers and Lipscomb, 2011). Normalisation of blood ammonia values is also used as an indication of the efficacy of hepatic encephalopathy therapy. However, it should be noted that there is not a perfect correlation between the presence of hepatic encephalopathy in cats with portosystemic shunts and hyperammonaemia. Thus, a normal blood ammonia level does not rule out the presence of hepatic encephalopathy. Determination of pre- and post-prandial total serum bile acid levels is more sensitive and specific as an indication of the presence of shunts in cats than blood ammonia levels, while demonstration of the shunting vessels by imaging is the gold standard for diagnosis.

Decreased metabolism

Primary urea cycle defects

Urea cycle disorders are total or partial defects in enzymes or transporters involved in the urea cycle (Lopes et al, 2022). The major urea cycle deficiencies in humans are N-acetylglutamate synthase deficiency, carbamoyl phosphate synthetase-1 deficiency, ornithine transcarbamylase deficiency, argininosuccinate synthase deficiency, argininosuccinate lyase deficiency, arginase-1 deficiency (or hyperargininaemia), ornithine transporter deficiency and citrin transporter deficiency (or citrullinemia type 2) (Figure 1). In humans, all deficiencies are autosomal recessive except for ornithine transcarbamylase deficiency, which is X-linked. Definitive diagnosis of these disorders is by determination of enzyme activity or genetic mutational analysis. However, plasma amino acid analysis in combination with evaluation of urine orotic and organic acids can permit a tentative diagnosis (Figure 2). Patients with low citrulline and high urinary orotic acid levels have ornithine transcarbamylase deficiency, while those with low citrulline and low urinary orotic acid levels have carbamoyl phosphate synthetase-1 or N-acetylglutamate synthase deficiency. Patients with high citrulline and high methionine levels have citrin transporter deficiency. Patients with high citrulline and low arginine levels with no argininosuccinate activity have argininosuccinate synthase deficiency, while these same patients with high argininosuccinate synthase activity have arginine succinate lyase deficiency (Auron and Brophy, 2012; Häberle, 2020).

A single case of a urea cycle abnormality in an 18-month-old cat has been reported. The cat was evaluated for stunted growth and post-prandial depression. Fasting blood ammonia level was 396 mcg/dl. The cat had low citrulline and high urinary orotic acid levels typical for ornithine transcarbamylase deficiency. No confirmation with determination of hepatic enzymatic activity was made (Washizu et al, 2004). Partial defects in urea cycle enzymes are present in humans. These partial defects can become unmasked with infection or severe malnutrition. This may have been in the case in a report of a 5-year-old castrated male Persian cat with refractory inflammatory bowel disease that presented with neurological signs and hyperammonaemia (286 µmol/litre). On amino acid analysis, the cat had low levels of citrulline, arginine and ornithine with normal urinary orotic acid levels. These results may suggest N-acetyleglutamate synthase deficiency or carbamoyl phosphate synthetase-1 deficiency (Dor et al, 2018).

Secondary inhibition of the urea cycle

Cats are sensitive to the development of hyperammonaemia from arginine deficiency. After an overnight fast, a single meal lacking this amino acid led to severe hyperammonaemia in kittens, with accompanying neurological signs and death (Morris and Rogers, 1978; Morris, 2002). Substitution of ornithine or citrulline in the arginine-free diet prevents hyperammonaemia. As obligate carnivores, cats cannot down-regulate the normally high activity of their amino acid catabolic enzymes during fasting. As a result, when fasted cats are fed a meal, ingested amino acids are rapidly deaminated as a source of energy, which causes a rapid increase in ammonia production. In other animals, this excess ammonia is efficiently converted into urea in the urea cycle. Arginine is an essential amino acid in cats necessary to generate ornithine, which in turn is necessary for synthesis of the key intermediate in the urea cycle, citrulline. However, because fasting results in low levels of arginine, this leads to a failure of the urea cycle resulting in hyperammonaemia.

Cobalamin is an essential cofactor for two mammalian enzyme systems, methionine synthase and methylmalonyl coenzyme A mutase. Cobalamin deficiency results in increased levels of methylmalonic acid, which indirectly inhibits carbamoyl phosphate synthetase-1, a rate-limiting enzyme in the urea cycle. Cobalamin deficiency has been reported in cats (Vaden et al, 1992; Simpson et al, 2012; Watanabe et al, 2012; Worhunsky et al, 2013) and was attributed to severe intestinal malabsorption from intestinal lymphoma or inflammatory bowel disease, exocrine pancreatic insufficiency or malnutrition (Simpson et al, 2012; Watanabe et al, 2012; Worhunsky et al, 2013). Cobalamin deficiency manifests clinically as a neurological disorder. These signs are, at least in part, related to the development of hyperammonaemia, although other neuropathological effects of hypocobalaminaemia are probable. Diagnosis is achieved by finding low levels of serum cobalamin and increased levels of serum methylmalonic acid in the sample.

Severe liver failure

Acute liver failure in the cat because of idiopathic hepatic lipidosis, suppurative cholecystitis/cholangitis, infectious causes and drug toxicity have all caused hyperammonaemia in the author's experience. Because the liver has a large reserve capacity for the conversion of ammonia into urea, the presence of hyperammonaemia is a relatively insensitive marker of liver function.

Other potential causes of decreased metabolism

Several disorders of fatty acid oxidation can lead to hyperammonaemia in humans (Ravindranath and Sarma, 2022). This hyperammonaemia is accompanied by lactic acidosis, increased liver enzymes, ketosis and/or hypoglycaemia. These fatty acid oxidation disorders have not been reported in the cat.

Increased ammonia production

Tonic-clonic seizures can lead to excess ammonia production in the muscles. During the intense muscle activity that accompanies seizures, muscle cells generate ammonia when adenosine monophosphate, produced by myokinase during adenosine triphosphate production, is degraded by adenosine monophosphate deaminase in the purine nucleotide cycle inside the muscle cells (Table 1; Hung et al, 2011; Nakamura et al, 2013; Karim et al, 2020). In humans, this post-seizure hyperammonaemia is transient, usually resolving within 8 hours. A study in cats demonstrated the occurrence of hyperammonaemia in 10/36 cats after tonic-clonic seizures (Nilsson et al, 2021). The hyperammonaemia resolved in all cases from 2 hours to 3 days post-seizure event. Of note, the determination of blood ammonia levels in this study used an in-house machine for which blood ammonia determination was validated in the dog but not the cat. Studies in humans have demonstrated an association between transient hyperammonaemia after a seizure and postictal confusion (Liu et al, 2011; Sato et al, 2016). Whether there is a connection in veterinary patients has not been explored. Since the increases in ammonia levels are transient, they may not need therapeutic intervention.

Urease-producing bacteria can hydrolyse ammonia in the urine to ammonium, which can then be excreted in the urine. However, as the urine becomes more alkaline as a result of the accumulation of ammonium, the lipophilic ammonia content in the urine increases. Ammonia easily diffuses into the vesical venous plexus and enters the systemic circulation. In the presence of a concurrent urinary obstruction (mechanical or functional interference with emptying), the transfer of ammonia to the vesical venous plexus and perivesical circulation is facilitated. Since this venous blood bypasses the portal circulation and drains into the inferior vena cava, hyperammonaemia can develop. Hyperammonaemia associated with a urinary infection with concurrent urethral obstruction was reported in a dog (Hall et al, 1987), but currently there are no reports of this occurring in the cat. Several bacteria are capable of producing urease, but among these, Corynebacterium urealyticum is noteworthy as it not only produces urease but can also be associated with an obstructive uropathy as a result of the development of an encrusting cystitis (Bailiff et al, 2005; Cavana et al, 2008; Briscoe et al, 2010; Maurey et al, 2019). The author has seen hyperammonaemia develop in a cat with concurrent C. urealyticum infection and urinary obstruction as a result of encrusting cystitis.

Undetermined aetiology

Hyperammonaemia associated with renal azotaemia is documented in two reports (Adagra and Foster, 2015; Carvalho et al, 2021). In the first study, four cats (three with stage 4 chronic kidney disease and one with an acute kidney injury) had elevated blood ammonia levels accompanied by the presence of mild (generalised weakness) to severe (recumbent, semi-comatose) neurological signs (Adagra and Foster, 2015). In the second study, hyperammonaemia was documented in 4/18 (22%) cats with renal azotaemia and was positively correlated with serum urea nitrogen, creatinine and phosphorus levels (Carvalho et al, 2021). Cats with an acute component to their kidney disease had the highest blood ammonia levels, likely reflecting more severe azotaemia in this group.

Hyperammonaemia in cats with kidney disease could be as a result of decreased elimination owing to disruption of the hepatic urea cycle or increased ammonia load. Little is known about the regulation of the urea cycle enzymes in cats. Increased protein (ammonia) loading in cats with renal azotaemia could occur with the feeding of high-protein diets, gastrointestinal bleeding, infections with urease-producing bacteria or excessive protein catabolism. Renal ammoniagenesis in response to acute or chronic metabolic acidosis or hypokalaemia may also contribute to hyperammonaemia. Although the bulk of the ammonia produced by the kidney is excreted in the urine, there is some spillover into the systemic circulation. Hyperammonaemia in azotaemic cats could also result from alterations in the gut microbiome that lead to increased gastrointestinal ammonia production and/or absorption. In humans with kidney disease, decreased renal excretion of urea leads to increased secretion of urea into the gastrointestinal tract, which is converted by urease-producing bacteria to ammonia. Studies in uraemic humans with kidney disease show that there are changes in the gastrointestinal environment, which result in decreased microbiome diversity and dysbiosis that could be associated with increases in ammonia production or absorption (Vaziri et al, 2013; Wong et al, 2014). The influence of these factors in cats with renal azotaemia is currently under investigation. Whether there is value in normalising blood ammonia levels (either acutely or chronically) in cats with kidney injury is unknown, and additional studies assessing the correlation of azotaemia and hyperammonaemia in cats are needed.

Treatment of hyperammonaemia in the cat

Treatment of hyperammonaemia depends partly on the aetiology (Table 2). It may involve dietary manipulation (normalisation of protein intake, alterations in dietary protein quality or replenishment of micronutrients such as vitamin B₁₂ or arginine) and/or medications to either alter the generation of ammonia in the gastrointestinal tract or bladder, or to scavenge circulating ammonia.

Cats with congenital portosystemic shunts often have dietary protein modulation incorporated into their clinical management. Typically, factors that help control hepatic encephalopathy in dogs and humans, such as limiting any excess protein in the diet combined with feeding dairy, soy and vegetable-based proteins, are applied to cats. However, almost nothing is known about whether this is the appropriate strategy in an animal that is an obligate carnivore. While considering this strategy, it is important to note that restriction of arginine can be associated with hyperammonaemia in the cat. However, parenteral cobalamin administration can ameliorate the neurological signs associated with this deficiency (Table 2) (Simpson et al, 2012).

Lactulose is often used to treat hyperammonaemia. In dogs and humans, lactulose works by both acidifying the colon and trapping ammonia in the form of ammonium, which is then excreted in the faeces, and modulating the bacterial flora in the colon to a less ammonia-generating phenotype. In humans, it also appears to promote the proliferation of bacteria in the small intestine, which then consumes ammonia in the process of protein production (Levitt and Levitt, 2019). Lactulose is effective in treating dogs and humans with hyperammonaemia as a result of liver disease or portosystemic shunts (Levitt and Levitt, 2019; Serrano et al, 2022). Although its efficacy has not been as well demonstrated in clinical studies in cats, the use of lactulose to treat hyperammonemia in cats with congenital portosystemic shunts is recommended (Tivers and Lipscomb, 2011). There is also the added difficulty of administering a sugary solution to a species that lacks the taste receptor for sweet.

In humans, the action of lactulose in managing hepatic encephalopathy is synergistic with the use of antibiotics (Pirotte et al, 1974; Mohammad et al, 2012; Sharma et al, 2013; Levitt and Levitt, 2019). Rifaximin is the antibiotic of choice in humans, but currently is too expensive to use in veterinary medicine. Instead, metronidazole or amoxicillin are substituted. In refractory cases, increasing dietary fibre to help further acidify the colon or altering the microbiome with probiotics may be of some additional benefit in controlling hepatic encephalopathy.

Several conditions can precipitate or promote hepatic encephalopathy, including infections, dehydration, electrolyte or acid–base abnormalities (especially metabolic alkalosis and hypokalaemia which promote renal ammoniagenesis) and gastrointestinal bleeding. Drugs aimed at ammonia scavenging either enhance the urea cycle in the liver or augment glutamine synthesis in peripheral tissues – particularly skeletal muscle (Häberle, 2020; Lopes et al, 2022). These drugs are currently primarily used in human medicine to treat inborn errors of urea synthesis. L-ornithine-l-aspartate, a stable salt of the two amino acids, is used as an ammonia scavenger. Ornithine, a urea cycle intermediate, activates the urea cycle enzyme carbamoyl phosphate synthetase, and as such can increase the capacity to detoxify ammonia. L-ornithine-l-aspartate is also a substrate for transamination reactions resulting in the increased synthesis of glutamate, leading to increased production of glutamine (and thus consumption of ammonia) in hepatocytes, skeletal muscle and brain. Sodium benzoate and sodium phenylacetate are additional ammonia scavengers. Sodium phenylacetate combines with glutamine to produce phenylacetylglutamine, which is excreted in the urine. Sodium benzoate combines with glycine to produce hippuric acid, which is excreted in the urine, and glycine is further replaced by synthesis, thus removing more waste nitrogen. There is no information on the use of these nitrogen scavenging drugs in cats.

Haemodialysis is used in humans to treat life-threatening hyperammonaemia. A single study in dogs describes the successful use of therapeutic plasma exchange to treat canine hyperammonaemia (Culler et al, 2020). There are no reports of its use in cats.

Conclusions

Hyperammonaemia should be suspected in cats presenting with signs of a metabolic encephalopathy. Blood ammonia concentration ideally should be done in-house with careful attention to sample collection and handling. Additional diagnostic testing may involve determination of total serum bile acid levels or diagnostic imaging to look for the presence of portosystemic shunting (congenital or acquired). Determination of indices of hepatic (serum liver enzymes and bilirubin) and kidney (creatinine) function, serum cobalamin and methylmalonic acid levels and in certain circumstances, plasma amino acid analysis and urinalysis for organic acids may be necessary for a definitive diagnosis. Treatment is either aimed at correction of dietary deficiencies, or at limiting protein degradation in the intestine and interfering with ammonia absorption in the intestine using diets with lower levels of protein, combined with the administration of lactulose and antibiotic therapy to modulate the production of ammonia.

KEY POINTS

• Blood ammonia testing should be considered in any cat presenting with signs of a metabolic encephalopathy such as seizures, altered mentation or cortical blindness.
• The most common cause of hyperammonaemia in the cat is congenital portosystemic shunting, which may be accompanied by failure to thrive, ptyalism and the presence of copper-coloured irises.
• Samples for blood ammonia determination should be free of haemolysis and kept cold. Plasma should be immediately separated, preferably via refrigerated centrifugation, and then assayed within the hour.
• The diagnostic work up for cats with hyperammonaemia not associated with portosystemic shunting should include investigation of hepatic and kidney function, whole body cobalamin status, and if necessary, determination of plasma amino acids and urinary organic acids.