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Bilirubin and porphyrins

18 min

Skills you'll build4 skills
  • Match each bilirubin fraction to a step in production, conjugation, or excretion
  • Explain a persistent direct-reacting bilirubin during recovery
  • Check light protection and interference before releasing a bilirubin result
  • Choose the first porphyria test for acute attacks, blistering, or painful photosensitivity

At the bench: Assess specimen interference before reporting a chemistry result

Try first

Try first

An adult has a total bilirubin of 4.8 mg/dL and a direct bilirubin of 0.4 mg/dL. Urine bilirubin is negative, and aminotransferases and alkaline phosphatase are within their intervals. At which stage of bilirubin metabolism is the excess accumulating?

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Bilirubin metabolism and fractions

Macrophages turn heme into unconjugated bilirubin, which is carried on albumin to the liver. Hepatocytes conjugate it with glucuronic acid, and the conjugated form is excreted in bile.1 The reported fractions depend on the method:1

  • Total bilirubin is measured with an accelerator that frees albumin-bound unconjugated bilirubin.
  • Direct bilirubin is the fraction that reacts without it. It is mostly conjugated bilirubin, plus delta bilirubin and a little unconjugated bilirubin.
  • Indirect bilirubin is total minus direct. It includes the error of both measurements.

An unconjugated-predominant rise points to increased production or reduced uptake or conjugation. A direct-predominant rise points to hepatocellular injury or cholestasis. Only conjugated bilirubin that circulates free reaches urine.2

Delta bilirubin

In prolonged conjugated hyperbilirubinemia, some conjugated bilirubin binds covalently to albumin. This delta bilirubin reacts as direct bilirubin, clears only as albumin turns over, and cannot be filtered into urine. During recovery, direct bilirubin can stay raised with urine bilirubin negative.3

Protect bilirubin from light

Light breaks bilirubin down after collection, so an exposed specimen reads falsely low. Separate the serum promptly and protect it in an amber container or other cover.4 A repeat on the exposed tube measures the same loss.

Choose the porphyria test by presentation

A generic porphyrin order can leave out the precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Liver disease, kidney disease, alcohol and medications also raise porphyrins.5

PresentationFirst tests
Current acute neurovisceral symptomsRandom urine PBG, ALA and creatinine
Blistering photosensitivityPlasma fluorescence scan with urine porphyrin fractions
Painful photosensitivity without blistersErythrocyte protoporphyrin, metal-free and zinc

A PBG more than 5 times the upper reference limit strongly supports an acute hepatic porphyria. Cover every porphyria specimen from light at collection.5,6

Follow the bench demonstration

Sources for this step
References
  1. Guerra Ruiz AR, Crespo J, López Martínez RM, et al. Measurement and clinical usefulness of bilirubin in liver disease. Adv Lab Med. 2021;2(3):352-361. doi:10.1515/almed-2021-0047
  2. Kwo PY, Cohen SM, Lim JK. ACG clinical guideline: evaluation of abnormal liver chemistries. Am J Gastroenterol. 2017;112(1):18-35. doi:10.1038/ajg.2016.517
  3. Zhao D, Wu GY. Prolonged direct hyperbilirubinemia following acute hepatitis: when not to worry? J Clin Transl Hepatol. 2024;12(12):985-987. doi:10.14218/JCTH.2024.00265
  4. Mayo Clinic Laboratories. Bilirubin, serum. Accessed August 29, 2026. https://www.mayocliniclabs.com/test-catalog/overview/8452
  5. Aarsand AK, To-Figueras J, Whatley S, Sandberg S, Schmitt C. Practical recommendations for biochemical and genetic diagnosis of the porphyrias. Liver Int. 2025;45(3):e16012. doi:10.1111/liv.16012
  6. Wang B, Bonkovsky HL, Lim JK, Balwani M. AGA clinical practice update on diagnosis and management of acute hepatic porphyrias: expert review. Gastroenterology. 2023;164(3):484-491. doi:10.1053/j.gastro.2022.11.034

Watch one

Read each decision and its explanation.

An adult had a common bile duct stone removed 3 weeks ago. The service asks whether the raised direct bilirubin means the duct is obstructed again. The urine was tested fresh, and the strip controls were acceptable.

What does the persistent direct bilirubin most likely represent?

TestResultPreviousReference intervalFlag
Total bilirubin3.1 mg/dL High3 weeks ago: 9.8 mg/dL9.8 mg/dL3 weeks ago0.3–1.2 mg/dL0.3–1.2High
Direct bilirubin2.7 mg/dL High3 weeks ago: 7.9 mg/dL7.9 mg/dL3 weeks ago0.0–0.3 mg/dL0.0–0.3High
Alkaline phosphatase148 U/L High3 weeks ago: 612 U/L612 U/L3 weeks ago40–129 U/L40–129High
Urine bilirubinNegative3 weeks ago: LargeLarge3 weeks agoNegativeNegative

Specimen: H 3, L 12, I 4. Serum separated promptly and protected from light; fresh urine

  1. Read the enzyme trend: alkaline phosphatase fell from 612 to 148 U/L.

    Alkaline phosphatase tracks bile flow, so its trend shows whether the obstruction was relieved.

  2. Read the bilirubin trend: total bilirubin fell from 9.8 to 3.1 mg/dL.

    Check the bilirubin trend next.

  3. Calculate the direct share: 2.7 ÷ 3.1 = 87% of the total, about the same as the 81% three weeks ago (7.9 ÷ 9.8).

    The direct fraction as a share of the total shows which fraction remains.

  4. Compare with the urine: a large urine bilirubin has become negative on a fresh specimen with acceptable controls.

    Free conjugated bilirubin reaches urine, and albumin-bound delta bilirubin cannot.

  5. Report the measured fractions. The pattern supports recovery with lingering delta bilirubin, and it cannot prove the illness has resolved.

    The routine direct result does not measure delta bilirubin separately.

The direct bilirubin of 2.7 mg/dL is most likely delta bilirubin clearing with albumin turnover, since alkaline phosphatase is falling and urine bilirubin is now negative.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A routine serum bilirubin specimen will wait before analysis. Which handling preserves the result?

Incorrect. Refrigeration is acceptable, but a clear tube leaves the serum exposed to light, and light can change bilirubin before analysis.

Correct. Reference-laboratory instructions require prompt separation and light protection, such as an amber container, for total and direct bilirubin. Storage limits follow the validated method.

Incorrect. Capping keeps the serum on the cells in a tube that light can reach. Separate bilirubin specimens promptly and protect them from light.

Hint
  1. Ask what breaks bilirubin down while the specimen waits.
  2. Cold storage does not shield a clear tube from light.

Review Bilirubin measurement and specimen quality

Problem 2 of 3 · Hints available

An acute hepatic porphyria is suspected in a patient with current acute neurovisceral symptoms. Which initial test panel is appropriate?

Incorrect. A generic porphyrin order can omit 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), the precursors that rise during an acute attack.

Incorrect. That pair is the first step for blistering photosensitivity. An acute neurovisceral attack is assessed through the porphyrin precursors.

Correct. Quantitative porphobilinogen (PBG) and 5-aminolevulinic acid (ALA) measure both precursors, and creatinine normalizes the random urine. Cover the specimen from light at collection.

Hint
  1. Match the first test to the presentation: an acute attack, blisters or painful light sensitivity.
  2. During an acute attack, the porphyrin precursors rise.
  3. A random urine needs a way to correct for how dilute it is.

Review Selecting porphyria tests

Problem 3 of 3 · On your own

An adult has a total bilirubin of 6.2 mg/dL and a direct bilirubin of 4.9 mg/dL. Urine bilirubin is positive, and alkaline phosphatase and gamma-glutamyl transferase are raised out of proportion to the aminotransferases. At which stage of bilirubin metabolism is the excess accumulating?

An indirect fraction of 1.3 mg/dL is a small share of the total. The increase is in the direct fraction.

Delta bilirubin is bound to albumin and cannot be filtered. Positive urine bilirubin shows free conjugated bilirubin.

Direct bilirubin is 79% of the total, urine bilirubin is positive, and the enzymes point to cholestasis. Conjugated bilirubin is backing up.

Hemolysis raises the unconjugated fraction and leaves urine bilirubin negative.

Review Bilirubin formation and fractions

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • A 55-year-old woman, MRN 7358210, is followed for cholestatic liver disease.
  • Her serum was separated on time into a clear tube and left uncovered on a bench beside a sunny window for 5 hours before testing.
  • QC is acceptable.
TestResultPreviousReference intervalFlag
Total bilirubin0.9 mg/dL Delta2 weeks ago: 2.6 mg/dL2.6 mg/dL2 weeks ago0.3–1.2 mg/dL0.3–1.2Delta
Alkaline phosphatase402 U/L High2 weeks ago: 395 U/L395 U/L2 weeks ago40–129 U/L40–129High
ALT58 U/L High2 weeks ago: 61 U/L61 U/L2 weeks ago7–45 U/L7–45High

Specimen: H 2, L 10, I 1. Serum in a clear tube, uncovered in daylight for 5 hours

Decision 1 of 3

What best explains the bilirubin of 0.9 mg/dL?

Five hours of daylight on a clear tube can lower bilirubin. The enzymes, which light does not affect, are at her usual values.

An improvement would usually bring alkaline phosphatase down as well. It is unchanged, and the tube sat in daylight for 5 hours.

Released bilirubin without checking light exposure

Light breaks down bilirubin after collection, so serum left uncovered or unseparated can read falsely low. Releasing the result without checking handling reports a bilirubin below the patient's true value, and repeating the test on the same exposed specimen reproduces the loss.

Delta bilirubin clears slowly with albumin turnover. A fall of this size in 2 weeks with unchanged enzymes points to a specimen problem.

Review Bilirubin measurement and specimen quality

Decision 2 of 3

What do you do with the bilirubin?

The result may be well below her true bilirubin, and a falsely low value would be interpreted as improvement.

The bilirubin that light destroyed is gone. A repeat on the exposed serum reproduces the loss.

Released bilirubin without checking light exposure

Light breaks down bilirubin after collection, so serum left uncovered or unseparated can read falsely low. Releasing the result without checking handling reports a bilirubin below the patient's true value, and repeating the test on the same exposed specimen reproduces the loss.

A comment leaves a falsely low number in the chart, where it can be trended as a real value.

A light-protected specimen gives a bilirubin that reflects the patient's true value, and the reason on the order explains the canceled result.

Review Bilirubin measurement and specimen quality

Decision 3 of 3

What do you do with the alkaline phosphatase and ALT from the same tube?

Light exposure changes bilirubin. The enzymes were measured on serum separated on time, and they are valid.

The serum was separated on time, and light does not break these enzymes down. Both are close to their previous values.

The redraw is for bilirubin. These results are valid from this tube, and a redraw would only delay them.

Review Specimen integrity and interference

The clear tube left in daylight explains a bilirubin fall that the unchanged enzymes do not support. Light breaks down bilirubin and does not affect the enzymes, so only the bilirubin is redrawn with light protection.

For reference

Sources checked