Biochemistry, Uroporphyrinogen

Fundamentals

In the human body, heme biosynthesis is mostly present in the bone marrow and liver tissue. 

• Bone Marrow - due to the large requirement for heme to sustain the production of hemoglobin, the bone marrow accounts for the majority of daily heme synthesis (over 80%). In erythroblasts and reticulocytes, heme synthesis gets regulated by the presence of the erythroid-specific enzyme aminolevulinate synthase 2 (ALAS2), which catalyzes the first step in heme synthesis. The gene for this enzyme has a promoter region with several erythroid-specific elements that are acted upon by erythroid transcription factors. Also, the ALAS2 mRNA has an iron-responsive element IRE- that leads to the upregulation of enzyme expression in the presence of iron.[2]
• Liver Tissue - in the liver, heme is utilized primarily as a prosthetic group for the various enzymes in the cytochrome P450 (CYP450) family; this accounts for the bulk of the remainder of heme synthesis in the human body. ALAS1 is the enzyme present in the liver that is responsible for the regulation of heme synthesis and operates with a feedback mechanism to maintain a relatively constant amount of heme for use.[3]

Therefore an interruption in any of the steps in the production of heme could result in the buildup of specific toxic heme precursors leading to a group of diseases called the porphyrias, which may present differently depending on the metabolite that accumulates and site of accumulation (hepatic vs. erythropoietic).

Cellular Level

There are eight enzymes involved in the biosynthesis of heme, and in cells, the first and last three steps of the pathway occur in the mitochondria while the rest occur in the cytosol. The production and metabolization of uroporphyrinogen III take place in the cytosol. 

Uroporphyrinogen III is one of the intermediates in the 8 step biosynthesis of heme. It forms from the linear compound hydroxymethylbilane, also called preuroporphyrinogen, by the cytosolic enzyme uroporphyrinogen III synthase (UROS). The newly cyclized compound is then converted to coproporphyrinogen III by the enzyme uroporphyrinogen decarboxylase (UROD).[4] However, in the presence of decreased UROS activity, hydroxymethylbilane may spontaneously close and form uroporphyrinogen I. Uroporphyrinogen I may then be acted upon by UROD to produce coproporphyrinogen I, which cannot undergo further metabolism, which results in the accumulation of the coproporphyrinogen I (a cytotoxic chemical), which can lead to clinically significant disease.[5]  Similarly, a deficiency or reduced activity of UROD can result in the accumulation of uroporphyrinogen III, which can then become oxidized to uroporphyrin and heptacarboxyl porphyrin. These are also toxic chemicals that can lead to sickness.[6]

Molecular Level

Uroporphyrinogens are cyclic tetrapyrroles (four pyrroles connected in a ring structure by methylene or methine bridges) with four propionic acid (P) groups and four acetic acid (A) groups. The various uroporphyrinogens differ by their arrangement of the side groups. The order of the side groups in uroporphyrinogen III is (in clockwise order) AP-AP-AP-PA, while the order in uroporphyrinogen I is AP-AP-AP-AP.[7]

Function

• Uroporphyrinogen III is a chemical intermediate in the biosynthesis of heme
• Uroporphyrinogen synthase is an essential enzyme in the production of uroporphyrinogen III; uroporphyrinogen decarboxylase is the enzyme involved in the metabolization of uroporphyrinogen III to further the process of heme production 
• Uroporphyrinogen I is a byproduct in heme biosynthesis and has no known useful function in humans- it forms in the presence of decreased UROS activity 

Pathophysiology

The group of diseases referred to as the porphyrias are metabolic disorders- they are caused by an abnormality in the function of the enzymes involved in the biosynthesis of heme. The anomaly usually manifests as an alteration in the activity (increased or decreased) of the enzyme, and most of the porphyrias have a genetic mutation that is responsible for the dysfunction. Porphyria cutanea tarda (PCT) is the lone exception, as it can develop in the absence of a gene mutation and is, in fact, most often caused by an acquired inhibitor (around 2/3 of cases).[8] Also, most genetic mutations occur in the gene that encodes the respective synthetic heme enzyme, with exceptions including rare instances of congenital erythropoietic porphyria (CEP), in which the causative mutation is in a transcription factor that controls the expression of the enzyme uroporphyrinogen III synthase.[9] Thus, any of the porphyrias may exhibit a predictable Mendelian genetic inheritance pattern as the genes for all the synthetic heme enzymes (or genes related to their expression) are found within the cellular genome (none of the genes are mitochondrial). 

The clinical presentation of the different porphyrias depends on the noxious metabolites that accumulate, their sites of accumulation, and the method by which those metabolites get excreted. Depending on the solubility of the compounds in different cellular components, they may accumulate in different areas of the body and be excreted by the liver (in bile and feces) or by the kidneys (in urine). This excretion is useful as levels of specific porphyrins can be measured in urine and feces to aid in the diagnosis. Also, certain symptoms such as cutaneous blistering or neurovisceral manifestations (abdominal pain, vomiting, weakness, psychiatric changes) are typically seen in certain porphyrias depending on the accumulation of porphyrins in specific areas such as the skin or nervous system. Knowledge of this can be used to help aid in forming the differential diagnosis of a patient with suspected porphyria.[10]  

Clinical Significance

The impaired metabolization of uroporphyrinogen III is the cause of two known porphyrias: congenital erythropoietic porphyria (CEP) and porphyria cutanea tarda (PCT). 

Congenital Erythropoietic Porphyria: Also known as Gunther disease, this porphyria is a rare autosomal recessive disorder caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS). In the presence of insufficient UROS activity, hydroxymethylbilane spontaneously closes and forms the non-physiological compound uroporphyrinogen I, which is then acted upon by uroporphyrinogen decarboxylase to form coproporphyrinogen I. Coproporphyrinogen I then oxidizes spontaneously to form the porphyrins coproporphyrin I and uroporphyrin I. In CEP, these toxic porphyrins mostly accumulate in RBC precursors in the bone marrow, as well as the teeth, bones, urine, and feces. They are not significantly elevated in the liver. This leads to disease characterized by severe cutaneous photosensitivity and hemolysis.[11]

• Cutaneous Photosensitivity - As the toxic porphyrins mentioned above accumulate in the bone marrow, they may be released into circulation and subsequently found in the plasma and mature erythrocytes. They then may travel to the skin, and upon exposure to light, generate free radicals that can damage cells and tissues. This results in skin friability and blistering in sun-exposed areas like the face, ears, neck, forearms, and hands. The bullae and vesicles that form may rupture, scar, and become infected. In severe instances, this can lead to skin mutilation and deformities.[12]
• Hemolysis - Due to the accumulation of porphyrins within erythrocytes, intravascular hemolysis is a major feature of CEP. In an effort to compensate, the bone marrow and spleen actually increase the production of heme and hemoglobin synthesis, although it is usually not enough to counter the amount of hemolysis/ineffective erythropoiesis. This leads to typical findings of hemolytic anemia with splenomegaly(extramedullary erythropoiesis), increased reticulocyte count, increased indirect bilirubin, and decreased haptoglobin. Blood smear typically reveals nucleated RBCs, basophilic stippling, anisocytosis, poikilocytosis, and polychromasia. In severe cases, the hemolysis may cause hydrops fetalis in utero or jaundice in the neonatal period. 

The extent of enzyme deficiency caused by the UROS mutation in each case is the major determinant of the age of onset and severity of symptoms seen in the disease. Treatment involves avoidance of exposure to sunlight, prompt treatment of skin infections, possible RBC transfusions with iron chelation, and in severe cases allogeneic hematopoietic cell transplantation. 

Porphyria Cutanea Tarda: PCT is the most common type of porphyria. It is due to a deficiency or decreased activity in the enzyme uroporphyrinogen decarboxylase (UROD) within the liver, and UROD activity less than 20% is usually required for the clinical manifestations of PCT to appear. A genetic mutation in the UROD gene can predispose one to the disease, as it will cause a decrease in UROD activity to 50%, but other various acquired factors are also needed. Therefore, penetrance is usually low in familial PCT, and there may not be an apparent family history of the disease. Most cases of PCT (~80%) in fact occur in the absence of a UROD mutation and are due exclusively to acquired factors causing a deficiency in UROD activity- these cases are referred to as "sporadic."[13] The decreased activity of UROD in the liver leads to the accumulation of uroporphyrinogen III, which is oxidized to uroporphyrin and heptacarboxyl porphyrin. These toxic porphyrins are transported from the liver to the skin and are excreted in urine and feces. This leads to the cutaneous symptoms characteristic of PCT. 

Decreased activity of UROD in the liver that is acquired environmentally or behaviorally is usually due to resultant iron accumulation or oxidative damage in hepatocytes. Thus, risk factors for iron accumulation in the liver such as inherited HFE mutations, or factors for liver damage such as alcohol abuse, smoking, and HCV infection are all implicated in the pathogenesis of PCT. Determining the presence or absence of these factors in a patient with suspected PCT is crucial as it can affect the management of the disease.[8]

PCT generally presents in an adult with chronic blistering, hyper or hypopigmentation, and scarring in sun-exposed areas of the body. The blistering after sun exposure is usually delayed, and therefore, many patients may not be aware that sunlight is causing their skin manifestations. Hepatic transaminase elevations and hepatomegaly are also characteristic in patients with PCT, reflecting the liver damage present in the disease. Treatment involves low-dose hydroxychloroquine and phlebotomy to decrease the iron deposition in the liver, and the elimination of susceptibility factors is also essential in the treatment of PCT.[14]