April 2010: Volume 36, Number 4
A Guide to Laboratory Assessment
By M. Laura Parnas, PhD, and Elizabeth L. Frank, PhD
A group of rare metabolic disorders, porphyrias are associated with defects in the enzymes involved in the heme or porphyrin biosynthetic pathway. Normally, the body makes the heme molecule needed for hemoglobin in a multi-step process. Porphyrins are produced during several steps of this process. However, individuals with porphyria lack certain enzymes in the pathway, causing abnormal amounts of porphyrins or related molecules to build up in the body and eventually be excreted. Each distinct porphyria has a characteristic accumulation pattern of heme precursors or reduced enzyme activities that can be detected readily in blood, urine, and feces. Individuals with even partial deficiencies of the enzymes in this pathway accumulate and excrete heme precursors and intermediates.
Clinically, patients with porphyria have either neurological complications or skin problems, although some individuals have both symptoms. These distinct disease manifestations have led clinicians to divide porphyrias into two subgroups: acute neurologic and non-acute cutaneous, according to the most dominant clinical manifestation. But porphyria symptoms mimic a variety of conditions, making correct diagnosis critical to successful management of the disease.
Here we describe how appropriately chosen and interpreted laboratory tests are sensitive and specific for the distinct porphyrias and play a crucial role in the diagnosis and management of these disorders.
Biochemistry: Heme Formation and Porphyrias
Biosynthesis of heme occurs in all nucleated cells, primarily in developing red cells of the bone marrow where hemoglobin is generated, and to a lesser extent in hepatocytes where heme-containing enzymes are produced. Enzymes generate the tetrapyrrolic heme structure in a series of steps that take place in two distinct cellular compartments: the first and the last three steps occur in the mitochondrion, and the intermediate steps take place in the cytosol (Figure 1).
Heme Biosynthetic Pathway
Click for Figure
Eight enzymes (red) catalyze synthesis of heme from glycine and succinyl-coenzyme A. Formation and polymerization of porphobilinogen (PBG) are followed by cyclization to uroporphyrinogen III, the octa-carboxylate (8 COO–) intermediate. Spontaneous cyclization to uroporphyrinogen I occurs to a lesser extent. Sequential decarboxylation to both tetra-carboxylate (4 COO–) coproporphyrinogen isomers occurs, although only the enzymatically-produced coproporphyrinogen III isomer can be metabolized by coproporphyrinogen oxidase to form the di-carboxylate (2 COO–) protoporphyrinogen. Oxidation to protoporphyrin IX and insertion of Fe2+ to form heme completes the biosynthetic cycle. Production of heme regulates the activity of ALA synthase, the rate-limiting enzyme in the pathway, by negative feedback inhibition (–).
Eight enzymes catalyze the formation of protoporphyrin IX and the chelation of iron to produce heme. The initial and rate-limiting reaction in the pathway is the condensation of glycine and succinyl CoA to form δ-aminolevulinic acid (ALA), which is catalyzed by δ-aminolevulinate synthase (ALAS). Porphobilinogen (PBG) synthase then catalyzes condensation of two molecules of ALA to generate the monopyrrole PBG. This step is followed by polymerization of four PBG molecules to form the linear tetrapyrrole hydroxymethylbilane. Hydroxymethylbilane synthase catalyzes this polymerization; however, spontaneous polymerization does occur if PBG concentrations are sufficiently increased.
Following polymerization, uroporphyrinogen III synthase catalyzes cyclization of the tetrapyrrole to form uroporphyrinogen III, although minor spontaneous cyclization occurs, generating the uroporphyrinogen I isomer. Then, uroporphyrinogen decarboxylase mediates the stepwise decarboxylation of uroporphyrinogen isomers to form coproporphyrinogen III and I. Only coproporphyrinogen III is oxidatively decarboxylated by coproporphyrinogen oxidase to protoporphyrinogen IX. Subsequent oxidation of this compound by protoporphyrinogen oxidase produces protoporphyrin IX, and finally ferrochelatase inserts the ferrous iron (Fe2+) to generate heme.
Under normal conditions, this highly efficient biosynthetic pathway enables most of the ALA produced to be converted to heme. Only minimal amounts of precursors and intermediates are accumulated and excreted, with the route of elimination largely directed by the inherent aqueous solubility of each compound. For example, the porphyrin precursors ALA and PBG both are water-soluble and chiefly excreted in urine. The octa-carboxylate intermediate uroporphyrinogen is also water-soluble and excreted renally. On the other hand, dicarboxylate protoporphyrin IX, which is water-insoluble, is excreted in feces via the biliary tract. The remaining porphyrin intermediates, including the tetra-carboxylate coproporphyrinogens, are somewhat water-soluble and appear in both urine and feces.
In the body, it is important to note that the cyclic tetrapyrrole intermediates exist in a reduced chemical state and are called porphyrinogens. Upon exposure to air, these compounds are oxidized rapidly to the corresponding porphyrins, the analytes measured in the clinical laboratory.
Genetic Defects in Porphyrias
Genetic defects in seven of the eight heme biosynthetic enzymes give rise to the porphyrias. Individuals inherit most of these diseases in an autosomal dominant (AD) fashion, with one functional copy of the gene present. Therefore, the enzyme deficiency is partial, with sufficient enzyme activity to maintain heme homeostasis. Nevertheless, pathway precursors and intermediates that precede the enzymatic defect accumulate in body fluids, causing characteristic signs and symptoms of the different porphyrias. Although each type of porphyria originates from a different genetic defect, the clinical manifestations of these disorders are similar, falling into either the acute or non-acute subcategories.
The four disorders comprising this group are: acute intermittent porphyria (AIP); variegate porphyria (VP); hereditary coproporphyria (HCP); and aminolevulinic acid dehydratase deficient porphyria (ADP). Each disorder is associated with a different defective enzyme (Table 1).
Click for Table 1
AIP is the most common of the acute porphyrias, and it is inherited in an AD manner, as are VP and HCP. In contrast, the extremely rare ADP is inherited as an autosomal recessive (AR) trait. The predominant clinical phenotype of these disorders is acute neurovisceral attacks, characterized by diffuse abdominal pain, peripheral neuropathies, and mental disturbances. These acute attacks commence during early adulthood, more frequently in women than men, and may be accompanied by skin lesions in VP and HCP.
The genetic abnormalities present in the three AD acute porphyrias produce approximately 50% of the normal enzymatic activity. The body maintains sufficient heme concentration as a result of upregulation of ALA synthase. Consequently, acute porphyrias have low clinical penetrance, with most individuals remaining asymptomatic for life.
However, approximately 20% of affected individuals manifest symptoms in the presence or absence of precipitating endogenous and exogenous factors. These exacerbating agents, including drugs, hormones, stress, and infection, increase demand on the liver for heme, which in turn induces heme synthesis. The enzyme deficiency becomes rate-limiting, causing precursors and intermediates to accumulate. Individuals with acute porphyrias experience variable incidence and severity of attacks that become more frequent upon exposure to precipitating factors. All patients with acute symptoms and some asymptomatic individuals have increased urinary excretion of PBG and ALA.
There are three non-acute porphyrias: porphyria cutanea tarda (PCT); erythropoietic protoporphyria (EPP); and congenital erythropoietic porphyria (CEP) (Table 1, above).
Among the non-acute porphyrias, PCT is the most common. Both PCT and EPP are inherited as AD traits, while the very rare CEP is inherited in an AR fashion. Clinically, these disorders are characterized by photosensitization of the skin due to accumulation of porphyrin intermediates in body tissues. The symptoms develop as a result of the light-absorbing properties of the porphyrin ring.
However, two distinct groups of manifestations occur with sun exposure, particularly on the hands, forearms, neck, and face. Burning, itching, and erythema that appear in childhood on sun-exposed areas are characteristic features of EPP. In this disease, a partial deficiency of ferrochelatase causes protoporphyrin to accumulate in red blood cells. In contrast, individuals with PCT and CEP typically experience increased fragility of the skin, with trauma leading to erosions, subepidermal bullae, hypertrichosis, and patchy pigmentation. Erosions and bullae heal slowly leaving scars and hyper- or hypo-pigmentation. Patients with CEP tend to have more severe symptoms that appear earlier. These individuals also may experience disfiguring mutilation of light-exposed body parts such as the nose, ears, and hands.
Because porphyrias are relatively rare, many clinicians may not be familiar with them. Furthermore, physicians may not suspect porphyria in their initial clinical workup because affected individuals present with ambiguous clinical phenotypes. In-depth understanding of the biochemical pathway of the disease, however, provides a logical guide for laboratory investigations.
Figure 2 (below) outlines a diagnostic algorithm for porphyria that incorporates clinical presentation with high suspicion of the disease. Since porphyrin precursors and intermediates are produced in excess and excreted in body fluids, initial testing should demonstrate abnormal elevation of porphyrins. If this is the case, interpretation of the specific pattern of precursor and intermediate compounds will lead to diagnosis. Additional investigation can be performed to identify the specific enzymatic defect and guide treatment.
Click for Figure 2
Dx: Patients with Acute Symptoms
For patients who present with acute manifestations, including diffuse abdominal pain, peripheral neuropathy, and/or mental disturbances, and for whom there is high suspicion for a porphyria attack, quantification of urinary PBG is the test of choice (Figure 2). While normal urinary PBG concentrations indicate that the patient is not suffering an acute attack currently, it does not completely rule out acute porphyria. If clinical suspicion for the disease remains high, testing should be repeated on a specimen collected when symptoms are present. Increased urinary excretion of PBG indicates presence of an acute porphyria, and further testing is necessary to identify the specific disorder.
Labs also use quantitation of porphyrins in feces to differentiate the acute porphyrias. No increase of fecal porphyrins indicates that AIP is the cause of the attack. Elevated fecal protoporphyrin and coproporphyrin suggest VP, while a significant increase of coproporphyrin is characteristic of HCP. VP can be confirmed by testing plasma or serum for porphyrin fluorescence; a characteristic peak at 626–628 nm confirms the diagnosis.
Dx: Patients with Non-acute Symptoms
For clinical manifestations of cutaneous photosensitivity that trigger suspicion of porphyria, the appropriate test is quantitative fractionation of urinary porphyrins (Figure 2). If urinary porphyrins are not increased, porphyria is unlikely. Excess urinary excretion of porphyrins, particularly uroporphyrin and heptacarboxylate porphyrin, along with the presence of the unique product, isocoproporphyrin, is diagnostic of PCT.
Slight or no increase in urinary porphyrin excretion when suspicion for non-acute porphyria is high, together with presentation during childhood, should also trigger evaluation of porphyrins in red blood cells (RBCs). Increased erythrocyte porphyrins are consistent with EPP.
Confirmation of the extremely rare CEP is also straightforward. These patients excrete massive amounts of uroporphyrin I and coproporphyrin I isomers, and they typically present with symptoms early in childhood. They also may have severe skin lesions.
Hepatoerythropoietic porphyria (HEP), a rare homozygous disorder of uroporphyrinogen decarboxylase, has also been described. Clinically, the disease resembles CEP, but can be distinguished by a predominance of protoporphyrin in blood and appearance of isocoproporphyrin in feces.
Enzyme and Molecular Testing
The ideal method to demonstrate the presence of a porphyria is testing for activity of the suspected defective enzyme; however, this is rarely necessary for diagnosis. In family studies, measuring enzyme activity is useful and it complements molecular testing for defective heme biosynthetic enzymes.
Labs can measure the activity of the cytoplasmic enzymes involved in heme biosynthesis in red blood cells; however, the mitochondrial enzymes require nucleated cells, such as fibroblasts or leukocytes. The only enzyme assay currently available for routine use measures hydroxymethylbilane synthase (HMBS) activity, the enzyme associated with AIP. In most patients with AIP HMBS activity in erythrocytes is reduced to approximately 50%, although significant overlap in values at the lower end of the reference interval exists between unaffected and affected individuals. Furthermore, there is a subtype of AIP in which the enzymatic defect is expressed in liver, but not in peripheral blood cells. Additional limitations of enzymatic testing are related to erythrocyte age, since younger RBCs have higher enzymatic activity, or to inappropriate specimen handling. Despite these limitations, determining HMBS activity helps confirm AIP and identify carriers in asymptomatic family members of affected individuals.
After biochemical analysis identifies the type of porphyria present, molecular testing can reveal the specific mutations causing disease. Most importantly, genetic testing aids in identifying carriers in families of symptomatic patients. Molecular testing cannot predict disease course or severity, but individuals carrying a mutation can be advised on lifestyle changes and ways to avoid precipitating agents and prevent symptoms.
Secondary Causes of Elevated Porphyrins
Abnormalities in metabolism and excretion of heme precursors and intermediates can also occur in the absence of inherited porphyrias. Such abnormalities can be caused by a variety of conditions, which should be considered at the time of interpretation of results in the context of clinical presentation.
For example, lead poisoning is one reason for elevated porphyrins in body fluids. It can cause attacks of acute abdominal pain and neurological disturbances that mimic acute porphyria attacks. Lead inhibits PBG synthase, the second enzyme in the pathway, and to a lesser extent coproporphyrinogen oxidase. As a result, lead toxicity is associated with significantly increased urine ALA concentration and increased urinary coproporphyrin, predominantly coproporphyrin III. Lead reduces intracellular iron availability, and zinc replaces iron as a substrate for ferrochelatase, forming zinc protoporphyrin (ZPP) in red blood cells. Tests for ALA, porphyrins, and ZPP provide indirect evidence for lead toxicity, but definitive diagnosis requires a finding of lead in blood and/or urine.
Labs sometimes encounter isolated increases in the concentration of urinary coproporphyrin during porphyria testing. These are typically associated with hepatobiliary malfunction that occurs in hepatitis, cirrhosis, and obstructive jaundice. In these conditions, coproporphyrin III is the predominant urinary isomer present. Inherited disorders of bilirubin metabolism—Dubin-Johnson syndrome, Rotor syndrome, and Gilbert disease— also increase urinary coproporphyrin concentrations; however, more coproporphyrin I than coproporphyrin III is excreted.
Treatment and Management
Clinicians treat acute attacks of porphyria by immediately withdrawing the suspected precipitating agents, treating co-existing illnesses and/or infections, and managing patients’ pain with non-porphyrinogenic agents. In some cases, drugs that trigger porphyria attacks must be replaced with alternative medications.
Today, intravenous heme preparations (hematin) are the preferred therapy for patients with a diagnosed acute porphyria attack. Hematin specifically inhibits hepatic ALAS activity and effectively decreases urinary PBG and ALA excretion. Additionally, carbohydrate loading in the form of oral or intravenous glucose can help halt an attack by moderate inhibition of hepatic ALAS activity. To prevent acute attacks, patients should: maintain adequate caloric intake, especially in the form of carbohydrates; avoid precipitating factors, including specific drugs, alcohol, and tobacco; and reduce stress.
Treatment of non-acute porphyrias is primarily preventive. In general, cutaneous symptoms can be minimized by avoiding ultraviolet light exposure and using topical sunscreens. For PCT, the treatment of choice is phlebotomy, which removes iron and stimulates erythropoiesis, resulting in decreased serum ferritin and urinary porphyrin excretion. An alternative treatment is administration of a low dose of chloroquine, which removes excess porphyrins from tissues. Patients should also avoid precipitants such as excess alcohol consumption, use of hormones, and smoking.
EPP patients also need to minimize sun exposure by wearing protective clothing and using topical sunscreens. Oral administration of β-carotene provides systemic photoprotection by quenching excited species formed by UV-activated porphyrins and preventing damage from oxidative radicals.
In the most severe form of porphyria, CEP, strictly avoiding sunlight and protecting skin from trauma are essential. Blood transfusions also help decrease hemolysis and suppress overproduction of porphyrins.
Improving Recognition and Diagnosis
Given the relatively uncommon occurrence of porphyrias, clinicians may initially suspect that patients presenting with neurological symptoms or skin problems have other, unrelated conditions. However, appropriate laboratory analysis can provide sensitive and specific diagnostic information. For example, urinary PBG should be the first test ordered for a patient with abdominal and neurological symptoms. Increased urinary excretion of PBG strongly suggests the presence of an acute porphyria. Additional biochemical testing can elucidate the specific type of acute porphyria.
In addition, adults experiencing cutaneous photosensitivity on sun exposure should be screened for urinary porphyrin excretion; a 24-hour specimen is preferred. The most common non-acute porphyria, PCT, can be diagnosed by its characteristic pattern of urine porphyrin excretion. Children who complain of burning, itchy skin following exposure to sunlight should be tested for erythrocyte protoporphyrin concentration. Enzymatic and molecular analyses, available from specialized reference laboratories, are best used to confirm a particular disorder and to identify family members at risk for the disease.
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M. Laura Parnas, PhD, is chief clinical chemistry fellow in the Department of Pathology at the University of Utah, Salt Lake City.
Elizabeth L. Frank, PhD, is medical director of Analytic Biochemistry and Calculi at ARUP Laboratories, and associate professor of pathology in the Department of Pathology at the University of Utah, Salt Lake City.