Hemoglobin, Iron, Bilirubin

What is hemoglobin, and where is it primarily synthesized in adults?

An oxygen-carrying protein, synthesized mainly in the bone marrow in adults.

What is the chain structure of hemoglobin?

4 globin chains — 2 alpha and 2 non-alpha.

How many heme groups does hemoglobin have, and how many oxygen molecules can it carry?

4 heme groups, each able to bind one oxygen molecule (total of 4 oxygen molecules).

Slide 4: Heme — Structure and Porphyria

What is heme composed of?

Chelation (binding) of iron to a porphyrin ring.

What is porphyrin made of?

4 pyrrole rings (C₄H₄NH).

What is porphyria, and what causes it?

A group of disorders caused by enzyme deficiencies in the heme synthesis pathway, leading to a buildup of porphyrin molecules.

Slide 6: Hemoglobin Clinical Significance

What are thalassemias?

Conditions involving decreased production of globin chains.

What are hemoglobinopathies?

Conditions involving abnormal globin chains.

Slide 7: Myoglobin

What is myoglobin structurally, and what is its function?

A heme/globin monomer that serves as an oxygen "storage" protein in skeletal and cardiac muscle.

What is the clinical significance of myoglobin as a cardiac marker?

It is the first cardiac marker to rise after an AMI (acute myocardial infarction), but it is not specific.

How quickly does myoglobin return to normal after an AMI?

It is cleared quickly, returning to normal in approximately 12 hours.

Slide 9: Iron — Distribution and Toxicity

Why is free iron a concern in the body?

Free iron is toxic.

How is iron distributed throughout the body?

In hemoglobin, storage iron, tissue enzymes, myoglobin, transport protein, and the labile pool; normally only a small quantity of iron is in plasma.

Slide 10: Iron Regulation

How much iron is absorbed from the diet per day?

1-2 mg/day.

How does the body conserve iron, and what is the normal route of iron loss?

There is no excretion mechanism; normal loss occurs through cell and blood loss.

What is hepcidin, where does it come from, and what does it do?

A protein produced by the liver that inhibits iron absorption.

Slide 11: Iron Storage

What is ferritin, and where is it found?

The main storage form of iron; found in nearly all cells, especially in the bone marrow, liver, and spleen; iron is readily available from ferritin.

What is hemosiderin, and how does it differ from ferritin?

Hemosiderin is partially degraded ferritin; iron stored in hemosiderin is not readily available.

Slide 12: Iron Clinical Significance

What are the causes of iron deficiency?

Blood loss, dietary deficiency, and malabsorption.

What is the eventual consequence of iron deficiency?

Anemia.

What genetic disease causes iron overload, and what is its underlying problem?

Hereditary hemochromatosis, caused by a problem with hepcidin.

Besides hereditary hemochromatosis, what else can cause iron overload?

Excess transfusions, leading to iron deposits in tissues.

Slide 13: Serum Iron Testing

What is the general method for measuring serum/plasma iron, and what steps are involved?

Lower the pH to release iron from transferrin, then form a complex with a chromogen for measurement.

What is an important pre-analytical consideration with serum iron testing?

Diurnal variation (iron levels vary throughout the day).

Slide 14: Iron Binding Capacity

What does iron binding capacity measure?

An indirect measurement of transferrin.

What are the two types of iron binding capacity?

Unsaturated iron binding capacity (UIBC) and Total iron binding capacity (TIBC).

What is the relationship between TIBC, UIBC, and serum iron?

TIBC = UIBC + serum iron concentration.

Slide 15–17: TIBC Testing

How is TIBC measured, and why is it considered a long, involved process?

Excess iron is added to saturate transferrin, unbound iron is separated and removed, and then the iron is tested; the process requires multiple steps including pH changes to separate iron from transferrin.

Slide 18–20: UIBC Testing

How is UIBC calculated?

A known amount of excess iron is added, free iron is measured, and: UIBC = iron added − free iron.

Using the example from the slides, if 15 µg/dL of iron is added and 7 µg/dL is measured as free iron, what is the UIBC and TIBC (given a serum iron of 10 µg/dL)?

UIBC = 15 − 7 = 8 µg/dL; TIBC = UIBC + serum iron = 8 + 10 µg/dL.

Slide 21: Transferrin Saturation

What does transferrin saturation (Tsat) measure?

The percentage of transferrin binding sites that are occupied by iron.

What are the two formulas for calculating transferrin saturation?

Tsat = (serum iron / TIBC) × 100, OR Tsat = [serum iron / (serum iron + UIBC)] × 100.

Slide 22: Transferrin Saturation — Examples

Using the example: serum iron = 78 µg/dL, TIBC = 302 µg/dL — what is the Tsat?

Tsat = (78 / 302) × 100 = 26%.

Using the example: serum iron = 68 µg/dL, UIBC = 237 µg/dL — what is the Tsat?

Tsat = [68 / (68 + 237)] × 100 = (68 / 305) × 100 = 22%.

Slide 23: Other Iron Tests

What is serum ferritin, how is it measured, and what does it reflect?

Measured by immunoassay; proportional to body iron stores.

What is serum transferrin concentration, how is it measured, and is it routinely tested?

Measured by immunoassay; not routinely tested.

What is serum hepcidin, how is it measured, and what is its current use?

Measured by immunoassay; currently used for research purposes only.

Slide 24: Iron Deficiency — Lab Results

In iron deficiency, what happens to serum iron, Tsat, transferrin concentration, TIBC, and ferritin?

Serum iron: decreased; Tsat: decreased; transferrin concentration: increased; TIBC: increased; ferritin: decreased.

Slide 25: Iron Overload — Lab Results

In iron overload, what happens to serum iron, Tsat, transferrin concentration, TIBC, and ferritin?

Serum iron: increased; Tsat: increased; transferrin: decreased; TIBC: decreased; ferritin: increased.

Slide 27: Hemoglobin Catabolism

What are the steps of hemoglobin catabolism from heme to bilirubin?

Heme and globin split → globin is broken down to amino acids → heme splits to iron and porphyrin → porphyrin is converted to biliverdin (green) → biliverdin is converted to bilirubin (yellow-orange).

Slide 28: Bilirubin Pathway

When bilirubin is first formed, what is it called, and what is its solubility?

Unconjugated bilirubin; it is insoluble in water.

How is unconjugated bilirubin transported to the liver?

It binds to albumin and is carried to the liver.

What does the liver do to bilirubin, and what does this accomplish?

The liver conjugates bilirubin with glucuronic acid, making it water soluble.

What happens to conjugated bilirubin after it leaves the liver?

It is excreted in bile into the intestine, where it is converted to urobilinogen, which is then converted to urobilin (brown — responsible for the color of stool).

Slide 31: Jaundice — Overview

What is jaundice (hyperbilirubinemia), and where does bilirubin deposit?

A condition of excess bilirubin in the blood; it deposits in the skin, mucous membranes, and sclera.

Is jaundice a disease?

No — it is a symptom of different diseases.

Slide 32: Pre-Hepatic Jaundice

What is the cause of pre-hepatic jaundice?

Hemolysis.

In pre-hepatic jaundice, what happens to plasma unconjugated and conjugated bilirubin levels?

Unconjugated bilirubin: increased (due to excess RBC breakdown producing more bilirubin than the liver can conjugate); conjugated bilirubin: normal.

In pre-hepatic jaundice, what are the urine bilirubin and urobilinogen results?

Urine bilirubin: negative (unconjugated bilirubin cannot pass into urine); urobilinogen: increased (more bilirubin reaching the intestine means more urobilinogen is produced and some is excreted in urine).

Slide 34: Hepatic Jaundice

What causes hepatic jaundice?

A liver problem — liver disease/damage or metabolic defects — causing bilirubin not to be processed normally (impaired uptake, processing, or movement).

In hepatic jaundice, what happens to plasma unconjugated and conjugated bilirubin levels?

Both unconjugated and conjugated bilirubin are increased, as the damaged liver cannot process or excrete bilirubin normally.

In hepatic jaundice, what are the urine bilirubin and urobilinogen results?

Urine bilirubin: positive (conjugated bilirubin leaks back into blood and into urine); urobilinogen: variable (may be increased or decreased depending on degree of liver damage).

Slide 36: Post-Hepatic Jaundice

What causes post-hepatic jaundice?

Obstruction (e.g., gallstones or tumor) that prevents bilirubin from leaving the liver.

What happens to stool color in post-hepatic jaundice, and why?

Stool becomes chalky and gray because urobilinogen is not formed in the intestine (conjugated bilirubin cannot reach the intestine to be converted).

In post-hepatic jaundice, what happens to plasma unconjugated and conjugated bilirubin levels?

Conjugated bilirubin: increased (backs up due to obstruction); unconjugated bilirubin: normal or slightly increased.

In post-hepatic jaundice, what are the urine bilirubin and urobilinogen results?

Urine bilirubin: positive (backed-up conjugated bilirubin spills into blood and urine); urobilinogen: absent/decreased (no bilirubin reaching the intestine to form urobilinogen).

Slide 38: Jaundice in Newborns

Why is jaundice dangerous in newborns?

Newborns have impaired bilirubin conjugation, and the breakdown of RBCs produces more bilirubin than the baby can process.

What is kernicterus, and what are its consequences?

Bilirubin deposits in the CNS, causing necrosis; it leads to irreversible damage and can be fatal.

Slide 39: Conjugated Bilirubin Measurement

What reaction is used to measure conjugated bilirubin, and what does it produce?

The diazo reaction: bilirubin + diazo sulfanilic acid → azobilirubin.

Why is conjugated bilirubin called "direct" bilirubin?

Because only conjugated bilirubin reacts directly with the diazo reagent without an accelerator.

Slide 40: Total Bilirubin Measurement

How is total bilirubin measured differently from conjugated bilirubin?

An accelerator is added to remove unconjugated bilirubin from albumin so it can also react with the diazo reagent: bilirubin + diazo sulfanilic acid + accelerator → azobilirubin.

What are two named variations of the total bilirubin diazo method?

Jendrassik-Grof and Malloy-Evelyn.

Slide 41: Unconjugated (Indirect) Bilirubin Measurement

How is unconjugated (indirect) bilirubin calculated, and why is it called "indirect"?

It is not measured directly; it is calculated as: Total bilirubin − conjugated bilirubin = unconjugated (indirect) bilirubin.

Slide 42: Bilirubin Measurement — Pre-Analytical and Reference Ranges

What is the critical pre-analytical requirement for bilirubin samples?

Bilirubin is light sensitive — always protect the sample from light.

What are the normal reference ranges for total, conjugated, and unconjugated bilirubin?

Total bilirubin: 0.0-1.5 mg/dL; conjugated (direct): 0.0-0.4 mg/dL; unconjugated (indirect): 0.0-1.1 mg/dL.

Slide 43: Direct vs. Indirect vs. Conjugated vs. Unconjugated

What is the relationship between direct, indirect, conjugated, and unconjugated bilirubin?

Total bilirubin = direct + indirect (OR conjugated + unconjugated); Direct = conjugated; Indirect = unconju