Heme Synthesis, Porphyrias, and Bilirubin

Pink plasma, a heme-positive urine with few red cells, and an icteric serum are all findings of heme metabolism. Laboratory evaluation of heme metabolism includes measurements of proteins, pathway precursors, and pigments. Plasma free hemoglobin indicates release from erythrocytes; urine 5-aminolevulinic acid and porphobilinogen reveal acute precursor accumulation; erythrocyte protoporphyrin supports selected porphyria evaluations; and serum bilirubin reflects heme breakdown and hepatic processing. Interpretation depends on the proper specimen and on knowing which part of the pathway the method measures.

Plasma free hemoglobin methods have assay-specific specimen, interference, and reporting rules. One current eight-wavelength method uses ethylenediaminetetraacetic acid plasma separated within 2 hours and reports total plasma hemoglobin or oxyhemoglobin according to the pigment pattern. Its limits and reference interval apply to that procedure.^{11 Mayo Clinic Laboratories. Plasma Free Hemoglobin, Plasma.} Serum myoglobin results are also method-specific. Its concentration reflects muscle release and renal clearance, and its broad tissue distribution limits etiologic interpretation.^{22 Mayo Clinic Laboratories. Myoglobin, Serum.} Current chest-pain guidance uses serial cardiac troponin I or T, with high-sensitivity assays preferred.^{33 Gulati M, et al. 2021 AHA/ACC guideline for the evaluation and diagnosis of chest… 2021.}

The detailed urine heme workflow appears in Urine Reagent-Strip Chemistry and Confirmatory Testing.

Each enzyme defect has a characteristic direction of change. The full pattern also depends on presentation and specimen.^{4,54 Belot A, et al. Update on heme biosynthesis, tissue-specific regulation, heme… 2024.5 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.}

Porphyrias are classified by the principal site of precursor overproduction and by their usual clinical pattern. Acute intermittent porphyria, hereditary coproporphyria, variegate porphyria, and ALA dehydratase deficiency porphyria are the acute hepatic porphyrias. Hereditary coproporphyria and variegate porphyria can also produce cutaneous disease. Porphyria cutanea tarda is usually sporadic. A minority of cases have familial uroporphyrinogen-decarboxylase deficiency. Erythropoietic protoporphyria, X-linked erythropoietic protoporphyria, and congenital erythropoietic porphyria arise mainly from erythroid precursor production. Biochemical patterns overlap, so the enzyme table guides test selection and cannot establish a subtype by itself.^{55 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.}

Quantitative porphyrin testing commonly uses high-performance liquid chromatography with fluorescence detection. Fecal porphyrins are extracted and fractionated chromatographically to separate coproporphyrin isomers and protoporphyrin. Erythrocyte hydroxymethylbilane synthase activity can support an acute intermittent porphyria or family evaluation. Overlapping activities limit its value as a standalone test. During evaluation of an acute neurovisceral attack, pair a total urine porphyrin result with urine ALA and PBG, because a porphyrin result does not include the precursors.^{55 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.}

During an acute attack, PBG and ALA usually rise substantially in acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria. A PBG result more than 5 times the upper reference limit strongly supports an acute hepatic porphyria in the appropriate setting. ALA dehydratase deficiency produces an ALA-predominant pattern. Lead exposure and hereditary tyrosinemia can also increase ALA. Properly handled PBG and ALA results within their reference intervals during current symptoms make an attack from acute intermittent porphyria, hereditary coproporphyria, or variegate porphyria unlikely. Also consider timing, renal function, recent hemin treatment, and specimen quality.^{5,65 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.6 Wang B, et al. AGA Clinical Practice Update on Diagnosis and Management of… 2023.}

Biochemical evidence comes before broad genetic testing in a symptomatic patient. A pathogenic variant can identify susceptibility in a relative or confirm the type after a diagnostic biochemical pattern. Increased precursors provide biochemical evidence. Attributing current symptoms to an acute attack also requires clinical assessment and, in patients with persistently high excretion, comparison with their baseline pattern.^{55 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.}

Specimen handling

Porphyria testing is vulnerable to preanalytic loss and dilution effects. Current international porphyria laboratory guidance recommends the following handling practices.^{55 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.}

1. Collect the symptom-appropriate specimen. Use a preservative-free random or morning urine for initial acute testing, and avoid a 24-hour collection.
2. Cover urine, plasma, blood, and fecal specimens immediately after collection, because porphyrins are light-sensitive.
3. Refrigerate or freeze according to the performing laboratory's instructions. PBG begins to decrease during room-temperature storage, and repeated freeze-thaw cycles can cause further loss.
4. Report urine PBG and ALA relative to creatinine. Interpret a very dilute specimen cautiously. A urine creatinine below 2 mmol/L, about 22.6 mg/dL, can produce a misleading ratio. Each laboratory sets its rejection criteria.
5. Follow the referral laboratory's requirements for pH, container, anticoagulant, transport time, and allowable storage. These conditions vary by analyte and method.

Lead and secondary porphyrin increases

Lead inhibits ALA dehydratase and ferrochelatase. ALA and erythrocyte protoporphyrin rise, and much of the excess erythrocyte protoporphyrin binds zinc. Zinc protoporphyrin (ZPP) also increases with iron deficiency and other disorders, and its sensitivity is poor at lower blood-lead concentrations. A normal ZPP does not exclude low-level lead exposure. A venous whole-blood lead measurement directly assesses exposure. Erythrocyte protoporphyrin testing can support the finding of disrupted heme synthesis.^{7,87 Centers for Disease Control and Prevention. Recommended Actions Based on Blood Lead Level. 2025.8 Mayo Clinic Laboratories. Protoporphyrins, Fractionation, Whole Blood.}

Liver disease, renal disease, alcohol exposure, medications, infection, toxins, and inherited bilirubin disorders can produce secondary porphyrin abnormalities. Fractionation across urine, plasma, erythrocytes, and feces helps separate these common secondary patterns from a porphyria. Clinical context and expert laboratory interpretation help evaluate small or isolated increases.^{55 Aarsand AK, et al. Practical recommendations for biochemical and genetic diagnosis… 2024.}

The direct result therefore differs from a chemically pure conjugated-bilirubin measurement. The subtraction and its limits appear in Core Chemistry Calculations.

Persistent delta bilirubin

During prolonged conjugated hyperbilirubinemia, some bilirubin becomes covalently attached to albumin as delta bilirubin. It reacts in the direct diazo assay and clears with albumin turnover, so direct bilirubin can remain increased after other liver chemistry results improve. Its albumin attachment prevents glomerular filtration, so delta bilirubin does not enter urine. Freely circulating conjugated bilirubin can enter urine.^{1010 Zhao D, et al. Prolonged direct hyperbilirubinemia following acute hepatitis. 2024.}

Persistent direct reactivity with absent urine bilirubin can therefore fit recovery from a documented cholestatic illness. Before attributing the pattern to delta bilirubin, review the trend, renal function, urine freshness, strip controls and interferences. The routine direct result does not separately quantify delta bilirubin. Report the measured fraction and communicate the supported interpretation. The pattern alone does not prove that ongoing liver or biliary disease has resolved.^{1010 Zhao D, et al. Prolonged direct hyperbilirubinemia following acute hepatitis. 2024.}

Fractionate an elevated total bilirubin, because the predominant fraction indicates the likely process. A conjugated or direct-predominant increase supports hepatocellular disease or cholestasis in most settings. An unconjugated-predominant increase supports increased bilirubin production or impaired uptake or conjugation.^{1515 Kwo PY, et al. ACG Clinical Guideline. 2017.} Urine patterns provide supporting evidence and vary with partial obstruction, urine stability, strip chemistry, and interfering substances. Detailed pad reactions and follow-up testing are covered in Urine Reagent-Strip Chemistry and Confirmatory Testing.

Quantitative manual urobilinogen methods use Ehrlich reagent, p-dimethylaminobenzaldehyde, to form a red product. Incomplete recovery limits precision, so many laboratories use a semiquantitative strip. Protect fresh urine from light, test promptly, and read the reaction within the method's stated window. Porphobilinogen, selected drugs, pigments, storage, and delayed reading can alter a result. Reference intervals and reaction limits are method-specific.^{99 Guerra Ruiz AR, et al. Measurement and clinical usefulness of bilirubin in liver… 2021.}

Selected cholestasis evaluations use serum bile acids with indication- and method-specific reference intervals. Fractionated bilirubin and the accompanying liver chemistries provide the general pigment pattern.^{99 Guerra Ruiz AR, et al. Measurement and clinical usefulness of bilirubin in liver… 2021.}

Inherited bilirubin patterns

Interpret an isolated bilirubin increase by its fraction and the supporting results. Normal aminotransferases and alkaline phosphatase, together with no evidence of hemolysis on the blood count, reticulocyte count, blood film, lactate dehydrogenase and haptoglobin, support an inherited processing pattern. They do not by themselves establish a genetic diagnosis.^{1616 Kyalwazi B. Bilirubin pathways and pitfalls. 2025.}

The report supplies the fraction pattern and relevant corroborating results for the clinical service. When an unconjugated increase comes with evidence of hemolysis, investigate the hemolysis before attributing the increase to an inherited conjugation disorder.^{1616 Kyalwazi B. Bilirubin pathways and pitfalls. 2025.} Describe an inherited pattern only after fractionation, and only when the supporting results agree.