Disorders of amino acid metabolism: Pathology review

A 6 month old infant girl named Joanna is brought to the emergency department by her mother. She’s concerned because, over the past couple of weeks, Joanna has been having repetitive episodes of sudden and rapid jerking movements associated with loss of consciousness. Upon physical examination, you notice that her sweat and urine has a musty odor, and that her head circumfernce is smaller compared with other babies of the same age and sex. Joanna’s mother mentions that she lives in a remote area and gave birth at home. Next to her, 17 year old Andreas comes in with left calf pain and swelling, which has been gradually increasing over the past few weeks. On further questioning, Andreas also mentions he has recently started to experience blurry vision, and has scheduled an appointment with his ophthalmologist. He has no history of immobilization, trauma or malignancy, and does not smoke or use recreational drugs. On physical examination, Andreas is unusually tall and thin, with long arms and legs, and long fingers. When you look into his eyes, you also notice that both his lenses have a partial dislocation down and inward. Okay, based on the initial presentation, both Joanna and Andreas seem to have some form of amino acid metabolism disorder. But first a bit of physiology real quick. Amino acids are the building blocks of proteins, and we have 20 of them. Now, all of them are made of a nitrogen group, a carbon skeleton, and a side chain that is unique to each amino acid. When amino acids are metabolized, the nitrogen is formed into a toxic compound called ammonia, which is sent to the liver. In liver cells, ammonia goes through a series of enzymatic reactions, known as the urea cycle, to be converted into the less toxic urea. Once urea is formed, it can go into the bloodstream and get excreted by the kidneys. Now, another way for liver cells to get rid of ammonia is to recycle it back to amino acids. For your exams, the most important recycling pathway involves pairing ammonia with alpha-ketoglutarate to form glutamate, which in turn combines with another ammonia molecule to make the amino acid glutamine. Now in cases of hyperammonemia, or elevated blood levels of ammonia, some of the excess ammonia may combine with alpha-ketoglutarate to form glutamate, which is the main excitatory neurotransmitter in the brain. Glutamate can then combine with another ammonia molecule to form glutamine, or with the help of vitamin B6, it can then get converted to GABA, which is the main inhibitory neurotransmitter in the brain. But since with hyperammonemia, there’s plenty of ammonia around, more glutamate will get converted to glutamine than to GABA. So, for your tests, note that this results in a buildup of glutamine, which is taken up by astrocytes, causing them to swell, as well as a decrease in GABA, which impairs neurotransmission. Over time though, the body’s pools of alpha-ketoglutarate will get depleted. The problem is that alpha-ketoglutarate is also a key intermediate of the Krebs cycle, also known as tricarboxylic acid cycle or TCA cycle for short. Now, remember that the Krebs cycle is one of the main cellular pathways to produce energy in the form of ATP, which is used for various cellular processes. One of them is ion transport by sodium- potassium pumps, which serve to pump sodium out of the cell, and potassium in. So as alpha-ketoglutarate levels fall, the Krebs cycle slows down, in turn reducing the production of ATP. As a consequence, the sodium- potassium pumps can’t do their job. This causes a build up of sodium ions in the cell, which allows water to flow into the cells via osmosis, leading to cellular swelling. For your exams, keep in mind that this primarily affects cells with high energy requirements like neurons, and the result is cerebral edema. The telltale sign of hyperammonemia is asterixis, which is a flapping tremor of the hand that appears when the wrist is extended, like a bird that’s flapping its wings. Additional signs and symptoms can include insomnia or hypersomnia, nausea, vomiting, mood changes, blurred vision, along with confusion, and even coma in some cases. Now, diagnosis of hyperammonemia mainly involves blood tests revealing increased ammonia levels, and the main treatment consists of a strict diet that limits protein consumption. For your exams, note that ammonia levels can be lowered with lactulose, which is a non-absorbable sugar, meaning it can’t be absorbed by the gastrointestinal tract. So once in the small intestine, lactulose gets broken down into lactic acid. This decreases the pH in the lumen, promoting the conversion of ammonia into ammonium ions. And ammonium anions can’t be reabsorbed, so they get excreted in the stool. Other treatment options include rifaximin or neomycin, which are antibiotics that kill ammonia- producing bacteria in the intestines. Other choices include benzoate, phenylbutyrate, or phenylacetate, which provide an alternative to the urea cycle, by combining with amino acids, like glycine or glutamine, and turning them into products that can be excreted in the urine. Now, hyperammonemia can occur either due to acquired or hereditary causes. A high yield acquired cause is chronic liver disease, where the liver isn’t able to remove ammonia from the blood, while hereditary causes include urea cycle defects, where a defect in an enzyme results in the overproduction of ammonia.

Okay, then! The most common urea cycle disorder is ornithine transcarbamylase deficiency, or OTC deficiency for short. This is caused by mutations in the OTC gene on the X chromosome. So, ornithine transcarbamylase deficiency is an X-linked recessive disorder, which means that all carrier males develop the disease, because they only have one X chromosome and thus one OTC gene available. On the other hand, females have two X chromosomes, so having a single mutation makes them a carrier, and two mutations are needed to have the disease.

Now, the OTC gene codes for an enzyme called ornithine transcarbamylase. Normally, ornithine transcarbamylase works in the urea cycle by combining ornithine with carbamoyl phosphate to form citrulline. So, deficiency of ornithine transcarbamylase results in an increase of carbamoyl phosphate in blood, which is then converted to orotic acid. Ultimately, this excess orotic acid gets excreted through urine, giving it a characteristic cloudy appearance. And the problem is that the orotic acid in urine can form crystals, which can obstruct the urinary tract. In addition, affected children can present with physical and mental developmental delay, along with failure to thrive.

If a test question mentions elevated orotic acid in urine, make sure you rule out orotic aciduria; this is an autosomal recessive disease that’s caused by a deficiency in the enzyme uridine monophosphate synthase, or UMPS for short. Orotic aciduria results in a defect in the pyrimidine synthesis pathway, leading to a decreased pyrimidine synthesis and an increase in orotic acid in the urine. One way to tell the two apart is that in orotic aciduria there is no hyperammonemia, and unlike OTC, it’s associated with megaloblastic anemia, which is a form of macrocytic anemia, with a mean corpuscular volume or MCV larger than 100 fL, and it's also characterized by the presence of megaloblasts. Megaloblastic anemia is caused by impaired DNA synthesis during red blood cell production in the bone marrow, which leads to continuing cell growth without division.

Diagnosis of ornithine transcarbamylase deficiency is done by genetic testing, looking for mutations in the OTC gene. Additional tests that can solidify the diagnosis include urinalysis revealing the presence of orotic acid in urine, as well as blood tests showing high orotic acid levels, hyperammonemia, high carbamoyl phosphate, and low citrulline.

Treatment includes ammonia-lowering medications, such as lactulose, rifaximin, neomycin, and benzoate, phenylbutyrate, or phenylacetate.

Next is phenylketonuria or PKU, which is an autosomal recessive disease, meaning that an individual needs to inherit two copies of the mutated gene, one from each parent, to develop the condition. Individuals affected by phenylketonuria have an impaired ability to use the amino acid phenylalanine. Normally, phenylalanine is processed into tyrosine by the enzyme phenylalanine hydroxylase with the help of a cofactor called tetrahydrobiopterin or BH4. Tyrosine is then made into several products including melanin, which is responsible for the pigmentation of skin, hair, and the iris of the eye.

Now, phenylketonuria can be divided into classic phenylketonuria, which occurs when there’s a deficiency in phenylalanine hydroxylase, and malignant phenylketonuria, which is caused by a deficiency in tetrahydrobiopterin. There’s also maternal phenylketonuria, which affects newborns whose mothers had untreated phenylketonuria during pregnancy. Mind that in maternal PKU, it is the mother and not the baby who actually has PKU. In all cases, phenylalanine can’t be broken down into tyrosine, causing melanin levels to decrease. At the same time, excess phenylalanine is broken down by other enzymes into several potentially harmful metabolites called phenylketones, including phenylacetate, phenyllactate, and phenylpyruvate. These end up being excreted in the urine and sweat. And that’s where the name phenylketonuria comes from! Another thing to note is that tetrahydrobiopterin also plays a role in the synthesis of neurotransmitters in the brain. Specifically, it’s a cofactor for tyrosine to be converted to L-dopa, which can then be converted to dopamine, as well as for tryptophan to be converted to 5-hydroxy-L-tryptophan, which can then be turned into serotonin. So, in malignant phenylketonuria, reduced levels of tetrahydrobiopterin will result in impaired neurotransmission in the brain.

Okay, so, symptoms of phenylketonuria usually present within the first few months of life and include a light skin tone and hair color, as well as blue eyes and a characteristic scent of urine and sweat. In a test question, that’s classically described as a “musty” or “mousy” odor. For unknown reasons, individuals with phenylketonuria also often present eczema, which is characterized by skin dryness, itchiness, and blistering. Neurological symptoms can also be present, including intellectual disability, abnormal gait, behavioral issues, and seizures. For your exams, keep in mind that these are going to be more severe in the case of malignant phenylketonuria. Finally, newborns with maternal phenylketonuria classically have microcephaly, or an undersized head, associated with intellectual disability, low birth weight, growth retardation, and congenital heart defects.

Okay, now, diagnosis of classic and malignant phenylketonuria, in many countries, is based on newborn screening to measure the blood levels of phenylalanine. For your exams, it’s important to remember that the blood sample is usually taken 2 to 3 days after birth. That’s because phenylalanine levels are typically normal right after birth due to circulating maternal phenylalanine hydroxylase.

Treatment should begin as early as possible and be maintained for life. It consists of a low phenylalanine and high tyrosine diet. What’s important to remember here is that the artificial sweetener aspartame contains phenylalanine and should be avoided. Sticking to this diet regimen is particularly important for individuals with phenylketonuria during pregnancy to prevent maternal phenylketonuria. And that’s a high yield fact! For those with malignant phenylketonuria, tetrahydrobiopterin supplements as well as L-dopa and 5-hydroxytryptophan administration will be also needed.

Moving on to maple syrup urine disease, this is an autosomal recessive disorder, in which the body cannot break down branched chain amino acids, so valine, leucine, and isoleucine. Normally, branched chain amino acids require special steps for their metabolism. First, the enzyme branched-chain amino transferase, or BCAT, converts them into branched- chain keto acids. Valine into alpha-ketoisovalerate, leucine into alpha-ketoisocaproate, and isoleucine into alpha-keto-beta-methylvalerate. And second, branched-chain alpha-keto acid dehydrogenase complex, or BCKD, turns these keto acids into isobutyryl-CoA, isovaleryl-CoA, and alpha-methylbutyryl-CoA respectively. For your test, keep in mind that BCKD needs a cofactor to work, which is thiamine or vitamin B1. And that's a very high yield fact!

Now, in maple syrup urine disease, there is a mutation in one of the genes that codes for the BCKD complex. Decreased BCKD complex activity means that all the branched chain amino acids and their branched- chain keto acids will build up in the blood. And some of these branched- chain keto acids use up other amino acids like aspartate, glutamine, and alanine, which are important for brain function and development, in order to get converted back into leucine, isoleucine, and valine. High levels of the branched- chain keto acid alpha- ketoisocaproate can also inhibit the Krebs cycle, slowing down the production of ATP. This impairs the function of sodium- potassium pumps, ultimately leading to cellular swelling and cerebral edema. At the same time, isoleucine is spontaneously converted into alloisoleucine, which can be then converted to sotolone, which has a very strong sweet smell. This molecule is excreted in the urine with the other metabolic products, giving the urine a distinct, sweet odor, and that’s why it’s called maple syrup urine disease!

Symptoms of maple syrup urine disease typically appear within 48 hours after birth, but they can also show up later in life. In a test question, look for sweet smelling urine, along with irritability, vomiting, poor feeding, and lethargy or sleepiness. In addition, some individuals may have intellectual disability. Another classic manifestation is opisthotonos, which is a severe simultaneous spasm of all muscles in the body, resulting in backward arching of the head, neck, and back. In some cases, these symptoms could be triggered by metabolic stressors like heavy exercise, infections, or fasting, where the body starts to break down its own proteins. For your exams, remember that if untreated, in seven to ten days, individuals can develop cerebral edema, seizures, coma, and respiratory failure can occur.

Diagnosis for maple syrup urine disease is based on blood tests that show elevated valine, leucine, isoleucine, and alloisoleucine in the blood, and urinalysis revealing increased alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, and alpha-ketoisovalerate.

The main treatment consists of a diet that limits consumption of valine, leucine, and isoleucine. Thiamine supplementation may also be beneficial.

Next is alkaptonuria. This is an autosomal recessive disorder caused by a mutation in the HGD gene coding for homogentisate oxidase. This enzyme normally catalyzes conversion of homogentisate to maleylacetoacetate, which is a step in the catabolism of tyrosine into acetoacetate and fumarate. So without homogentisate oxidase, homogentisic acid builds up in multiple organs and tissues, including the skin, connective tissue, ear cartilage, sclera, and articular cartilage. Signs and symptoms vary depending on the tissue affected. If that’s the skin, connective tissue, tympanic membranes, sclera, there can be bluish or black discoloration of these tissues, also known as ochronosis. For your exams, another extremely high yield finding is arthralgias or joint pain, which sometimes can interfere with activities of daily life. At the same time, remember that all that homogentisic acid will get excreted in the urine, so the telltale sign is black colored urine.