Hepatic Artery 

Portal Vein

Hepatocytes

Kupffee cells

Metabolism

Detoxificatoin 

Excretion/Secretion

Storage

Hydrolysis

Hydroxylation 

Oxidation/reduction

Carboxylation 

Demethylation

What are Cytochrome P450 enzymes? What is their function?

Heme & Unique apoproteins

Function: Drug removal by oxidation or demethylation, 3 phases of reactions inturn creating hydroxyl group (chemical now water-soluble)

Purpose is to make chemical water soluble for bile/urine excretion

• 7% of weight is glycogen
• 10% of total body content of iron is stored as ferritin
• Stores Fat soluble vitamins: A, D, E, K, B12
• Converts excess fatty acids to adipose tissue for long term storage

Trans- Insoluble in water

Cis- Soluble in water

1. Heme---> Biliverdin (Via Heme oxygenase, addition of O₂, release of Fe, CO)

2. Biliverdin---> Bilirubin (via Biliverdin reductase, NAD⁺/NADP⁺ is oxidized)

• It gets bound to albumin, circulates through the blood, delivered to the liver (unconjugated/indirect form)
• Dissociates at hepatocyte membrane 
• Ligands carry the bilirubin to the microsomes 
• Bilirubin is then rapidly conjugated w/glucuronic Acid by UDP gluconic acid 
• Now water soluble (conjugated/direct Bilirubin form)

Then gets excreted into bile, sm amnt reabsorbed by hepatocytes, transported to urine

Explain the process of excretion of bilirubin

1. Excreted into hepatic duct
2. Combined in gall bladder secretions via cystic duct
3. expelled through common bile duct into duodenum
4. hydrolyzed by B-glucuronidase (liver, intestine, bacteria)
5. Anaerobic degradation--> Urobilinogen
6. Most excreted through feces, (10-20% abs and recycled, sm amount secreted in urine)

yellowing of the skin, sclera, and mucous membranes mostly due to increased bilirubin concentrations

Can be caused by carotene and drugs 

increased production and release of bilirubin, mostly unconjugated, usually has to do with hemolytic or ineffective erythropoiesis.

Increased conjugated bilirubin may be found in the gut depending on the degree of hemolysis

• Hereditary Hemolytic Process
• Acquired Hemolytic process
• Ineffective Erythropoiesi
• Physiologic Jaundice of Newborn
• Impaired delivery of bili to the liver.

What are the two different types of hepatic jaundice?

Retention

Regurgitation

Transport defect of bilirubin to liver 

• Conjugated is <0.2 mg/dL
• Urine bili- neg
• Urine Urobilinogen- decreased or normal

Hepatocyte issue: damage or defect, or excretion impairment

Uptake, conjugation, and excretion impairment 

Increased: total bili, conj bili, urine bili, and urine bilinogen

• Increased total bili
• Cong/unconj bili vary by disorder and degree of damage
• Decreased urobilinogen in feces (clay colored white stool)

Physiologic Jaundice of Newborn

Gilbert Syndrome 

Criger-Najjar

Obstructive, blockage of flow of bie from liver into the intestine

Bile overflows back into circulation

Stools: clay white color due to no bilirubin in intestine and no urobilinogen being formed

Urine: large amount of bilirubin bc this is the only route of excretion 

Delta Bili: conjugated to albumin so it is not filtered by kidneys into the urine, so it would show a increase in plasma 

the liver may be damaged....resulting in decreased conjugation ability

Unconj bili may be increased due to decrease in conjugated bili.

(although rarely to the degree that conjugated bili is elevated)

>15 mg/dL after few days of birth

>10 mg/dL for more than 2 weeks after birth

Immature liver and hemolysis during birth 

Liver enzymes dont function until after birth...causing increase in bilirubin

Treatment: Phototherapt with monochromatic blue light to oxidize bilirubin into a soluble form for excretion

• Chronic/benign condition
• Regurgitation type hepatic jaundice
• Excretory impairment of bilirubin due to obstruction
• (Removal of bilirubin from hepatocyte  and excretion into bile is impaired)
• Total Bili: 2-5 mg/dL, over 50% conjugated bili, and delta bili seen

• Retention Hepatic Jaundice 
• Decrease or absent UDP transferase
• Type 1- No UDP, FATAL, Kernicturus, unconj bili 20-50 mg/dL
• Type 2- 10% activity of UDP trans. only, unconj ~20 mg/dL

• Retention Hepatic Jaundice 
• UDP transferase activity about 20-50%
• Unconjugated about 1-5 mg/dL

1. Bilirubin pigments react with Diazo Salt (reacts with conj)
2. Addition of Caffeine Benzoate (accelerator) unconjugated is dissociated from albumin 
3. Read at 600 nm for total bilirubin 

Conjugated only can be measure by skipping the accelerator step...subtract form total bili to get unconj.

What accelerator is used in the thin-film determination of bilirubin? What do you measure absorbance at?

Dyphylline

Measure at 540nm then 460nm to correct for spectral difference (conj and unconj)

When is direct spec used to measure bilirubin? How is it read?

used only on newborns <1 month old

Read at 455nm then 575nm

(Subtract second reading from first to correct for hb interference)

proportional to serum levels of bili since plasma of newborns does not contain any pigments that would interfere

HPLC 

High-Performance Liquid Chromatography

• Serum or plasma
• No hemolysis (deceases rxn with diazo)
• No Lipemia (interferes with spec)
• Keep out of natural light and fluorescent light ( dec 10% in 30 min)
• Fridge 7 days
• Freeze up to 3 months

Uroporphyrin

Coproporphyrin

Protoporphyrin

Uroporphyrin-->Coproporphyrin-->Protoporphyrin

Uroporphyrin has the greatest number of carboxyl groups

What are the Neurologic  types of porphyrias?

1. Acute intermittent 
2. Variegate
3. Coproporphyria

1. Congenital Erythropoietic 
2. Porphyria Cutanea Tarda
3. Protoporphyria

excess excretion of ALA and PBG 

(porphryin precursors)

Parathesia (skin tingles), weakness, inability to talk, paralysis, blindness, seizures

Abdominal pain, vomiting, constipation, fever, leukocytosis, hypertension

• Dec. Uroporphrinogen Synthase I
• Increased Urinary ALA
• VERY INCREASED URINE PBG
• Inc Liver Enzymes

• Increased Urine ALA
• Increased Urine PBG
• VERY INCREASED FECAL PORPHYRINS (Proto and Copro)

• Increased Urine Coproporphyrin
• Increased Urine ALA
• Increased Urine PBG

• excess porphyria production and excretion
• precursors are unaffected

What porphyria has an enzyme defect of

Uroporphyrinogen III cosynthase

• Red pigmented urine (increased excretion of coproporphyrin and uroporphyrin)
• Increased blood porphyrins

• No urine excretion of Porphyrins or precursors 
• Increased protoporphyrin IX in RBC, plasma and stool
• Increased fecal coproporphyrins and protoporphyrins
• Fluorescent fecal material

• Increased urinary uroporphyrin and coproporphyrin 
• Normal PBG and ALA

Liver Disease

Heavy Metal or organic solvent toxicity

due to the protein binding of lead (changes structure and function)

Inhibits the activity of PBG synthase and incorporation of iron into heme

• Chromogenic reaction and anodic stripping voltammetry (lack sensitivity)
• Atomic Absorption Spectrophotometry (most commonly used)

Chelation with EDTA

Monitor amount of urinary lead 

Removal of source 

• Watson-Shwartz (Ehrlichs Reagent--red color means positive)
• Confirmatory test- Hoesch Test (doesnt react with urobilinogen)

Ion exchange chromatography (Ehrlichs reagent)

(ALA uses cation column)

1. Porphyrins isolated, acidified, and extracted into organic solvent
2. seperated by solvent extration or chromatography
3. orange/red fluresence read at 620-630 nm

• Prolonged enzyme increase 
• HBsAg and Anti-HBc seen first
• Anti-HBs seen later 
• IgG/IgM

Anti-HCV (EIA)

(NAT-Viremia)

IgM Anti-HDV

IgA Anti-HDV

HDV Ag

RNA

Hep D 

incomplete RNA, needs Hep B to replicate