October 2022 – Biochemical Genetics

A 45-year-old Caucasian woman with a history of drug allergies (barbiturates and sulfonamides), depression, as well as recurrent acute episodes of generalized fatigue, dyspnea, and significant abdominal pain, was referred to Mayo Clinic for biochemical and molecular testing. Family history (Figure 1) revealed a maternal grandmother who died unexpectedly at the age of 36 after receiving barbiturates in preparation for an appendectomy. Even though no molecular investigations were performed, acute intermittent porphyria (AIP) was listed as the cause of death on the death certificate. Molecular genetic testing of the proband’s maternal aunt, however, was reportedly suggestive of variegate porphyria (VP) instead, while urinary porphyria testing (specimens not collected during attack) of the patient along with relevant family members were all negative and/or indeterminate (Table 1). Of note, multiple maternal family members including this aunt were reported to have symptoms and skin manifestations possibly consistent with lupus.

Despite the clinical presentation and family history of the patient, biochemical testing requested by the referring clinician only encompassed erythrocyte porphobilinogen deaminase (PBGD) activity as a means to establish/rule out AIP. No additional testing was ordered to investigate the possibility of VP and/or other acute hepatic porphyrias. Erythrocyte PBGD results were unremarkable (Table 1), and a subsequent molecular acute porphyria panel was reluctantly ordered by the clinician at the insistence of the patient. This later revealed that the patient was heterozygous for a well-known autosomal dominant pathogenic variant (c.503G>T, p.R168L) within the PPOX gene (Figure 2), which is consistent with a diagnosis of VP.

Figure 1: Family tree and relevant medical history
Figure 2: Sanger sequencing result for the PPOX gene
Table 1: Biochemical testing

Considering the clinical presentation and family history of this patient, which biochemical test/s should have been ordered for a more timely diagnosis prior to molecular testing?

  • Erythrocyte protoporphyrin fractionation; samples collected during an acute neurovisceral attack.
  • Urine (including porphobilinogen) and fecal porphyrins; samples collected during an acute neurovisceral attack.
  • Urinary porphobilinogen only; samples collected after an acute neurovisceral attack.
  • Erythrocyte porphobilinogen deaminase enzyme activity; samples collected during an acute neurovisceral attack.

The correct answer is ...

Urine (including porphobilinogen) and fecal porphyrins; samples collected during an acute neurovisceral attack.


Although the clinical presentation and medical history of the patient is consistent with an acute hepatic porphyria, inappropriate porphyrin testing and sample collection conditions impair the proper differentiation of acute porphyria subtypes, subsequently leading to a delay in and/or incorrect diagnosis. A timely differentiation of these subtypes is imperative, as some acute porphyrias such as VP and hereditary coproporphyria (HCP) have the propensity to lead to photosensitivity and ensuing cutaneous lesions. The following brief discussion of the aforementioned testing options are based on the Mayo Clinic acute porphyria testing algorithm,1 and depict some of the most important pitfalls and advantages to keep in mind when ordering biochemical testing for these diseases: 

OptionUrinary porphobilinogen (PBG) only; samples after an acute neurovisceral attack

While random porphobilinogen testing is valuable in the identification of an acute porphyria, PBG may be elevated in acute subtypes other than AIP, i.e., VP and HCP.2 This may be ascribed to the idea that aberrant accumulation of heme biosynthesis pathway intermediates activate delta-aminolevulinate synthase 1 enzyme activity, subsequently leading to induced elevations of PBG and aminolevulinic acid.3 Therefore, positive PBG measurements alone cannot be used for differentiation of acute subtypes, and additional testing is required for further classification.2 Furthermore, since porphobilinogen levels typically normalize within a few days after an acute neurovisceral episode, it is imperative that samples are collected during such an episode.4 This could explain the uninformative/normal urine porphyrin results obtained in the presented case and the relevant family members, as all specimens were reportedly collected in between attacks.

Option – Erythrocyte PBG enzyme activity; samples collected during an acute neurovisceral attack

PBGD catalyses the third enzymatic step of the heme biosynthesis pathway which encompasses the combination of four porphobilinogen molecules to form hydroxymethylbilane.3 Although a deficient PBGD enzyme activity is characteristic of AIP, unaffected erythrocyte activity does not rule out AIP, as 5%–10% of affected individuals only present with deficient activity in nonerythroid cells.5 A deficiency in this enzyme may also be masked by artificial stimulation via excessive alcohol ingestion,6 as well as by the collection of specimens during an attack.7 In addition, a normal PBGD activity alone will neither confirm nor rule out other acute hepatic porphyrias, subsequently being uninformative in the presented case. 

Option – Protoporphyrin fractionation; samples collected during an acute neurovisceral attack 

Erythrocyte protoporphyrins (metal free and zinc-complexed) concentrations are generally unaffected in acute porphyrias2 and are therefore not a relevant testing option in the current situation. 

Option – Urine (including porphobilinogen) and fecal porphyrins; samples collected during an acute neurovisceral attack

Besides the valuable contribution of PBG, urinary porphyrins may provide supplemental information about alternative porphyrin-intermediates (uroporphyrin to coproporphyrin) that could be auxiliary in the differentiation between acute porphyria subtypes. However, since VP and HCP present with nearly indistinguishable urinary profiles, fecal porphyrin testing is required to differentiate between these subtypes and is subsequently invaluable in the biochemical diagnosis of VP.7 Some of these characteristic/distinguishable findings are based on the observation that while the fecal coproporphyrin III/coproporphyrin I ratio is elevated in VP, it is even higher in HCP, and typically normal in AIP. Moreover, elevated fecal protoporphyrin excretion is seen only in VP cases and generally remains unaffected in other acute hepatic porphyrias. Consequently, fecal testing is a key component that should have been considered in the presented individual. However, as in the case of PBG measurements, the diagnostic success of these tests rely on sample collection timing to avoid false negatives.4,7


Based on some of these caveats, it is clear that no single biochemical test can differentiate between acute porphyria subtypes, and a comprehensive view of these (as described1) are required for an accurate and timely diagnosis of these patients.


  1. Mayo Clinic Laboratories (2022). Porphyria (acute) testing algorithm, https://www.mayocliniclabs.com/it-mmfiles/porphyria__acute__testing_algorithm.pdf
  2. Tortorelli S, White AL, Raymond K. Disorders of porphyrin metabolism. In: Biochemical and molecular basis of pediatric disease. 5th ed. PA: Academic Press; 2021: 503-528
  3. Bonkovsky HL, Guo JT, Hou W, Li T, Narang T, Thapar M. Porphyrin and heme metabolism and the porphyrias. Compr Physiol. 2013;3(1):365-401. doi:10.1002/cphy.c120006. PMID: 23720291
  4. Gounden V, Jialal I. Acute Porphyria. In StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. https://www.ncbi.nlm.nih.gov/books/NBK537352/
  5. Sassa S. The porphyrias. Photodermatol Photoimmunol Photomed. 2002;18(2):56-67. doi:10.1034/j.1600-0781.2002.180202
  6. McColl KE, Thompson GG, Moore MR, Goldberg A. Acute ethanol ingestion and haem biosynthesis in healthy subjects. Eur J Clin Invest. 1980;10:107-112. doi:10.1111/j.1365-2362.1980.tb02068
  7. Trier H, Krishnasamy VP, Kasi PM. Clinical manifestations and diagnostic challenges in acute porphyrias. Case Rep Hematol. 2013;628602. doi:10.1155/2013/628602. 

Zinandré (Zin) Stander, Ph.D.

Fellow, Clinical Biochemical Genetics
Mayo Clinic

Silvia Tortorelli, M.D., Ph.D.

Consultant, Laboratory Genetics and Genomics
Mayo Clinic

Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

Linda Hasadsri, M.D., Ph.D.

Consultant, Laboratory Genetics and Genomics
Mayo Clinic

Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

MCL Education

This post was developed by our Education and Technical Publications Team.