Lab 9 - Bilirubin Metabolism

Question 1

Br I

Answer 1

Indirect, bound to albumin, unconjugated, some get into blood

Question 2

BrII

Answer 2

Direct, bound to glucuronic acid=conjugated, a little amount get into blood

Question 3

Metabolism of bile acids

Answer 3

1️⃣Br+albumin in blood make brI and goes into liver
2️⃣br+glucuronic acid in liver make brII and is exreted into bile
3️⃣bacteria reduce br to urobilinofen which is either further red tot stercobilin or UBG is absorbed into blood(small molecule!) to the ileum
4️⃣from the ileum it will either be absorbed and go to the kidneys to be exreted, or to the liver for urea production

Question 4

serum bilirubin measurement. Normal value

Answer 4

diazo method: Spectrophotometroc method. With SERUM sample

Tot br: caffeine/alcohol + diaso + NaCl
Direct br: diaso + NaCl

Tot Br= 8 μmol/l, horse: < 40 μmol/l (bc no gallbladder for storage - circ storage instead!)

Question 5

Causes of incr Indirect br in the plasma

Answer 5

􏰀 acute haemolysis
􏰀 absorption of haemoglobin following massive internal haemorrhage, or after big
haematoma formation (so called ”resorption icterus”)
􏰀 transfusion of stored blood, which contains many dyeing or dead RBCs
􏰀 Decreased rate of conjugation of Br I by liver cells
􏰀 i Decreased uptake of Br I from the blood by liver cells

Question 6

Causes of incr Direct br in the plasma

Answer 6

(BrII) Ine Dykker Ikke
1️⃣Incr production after intravascular hemolysis
2️⃣decr excretion bc impaired hepatic function
3️⃣Impaired hepatic function due to obstruction of bile canal: swelling, inflammation, fibrosis, cells swell, blockage, compression

Question 7

Measurement of bilirubin in the urine

Answer 7

Gmelin test ((br)biliverdin, UBG)
Ehrlich test (UBG)

Diazo test strip (Br)

Question 8

🍏Gmelin test

Answer 8

Nitric acid has to be carefully layered under urine in a test tube, and the width of the differently coloured layers at the meeting of the two fluid phases has to be evaluated.

􏰂 condensed material on the surface of the glass tube - acidic urea, cats!
􏰂 yellow - urine itself
􏰂 white (opaque) – protein (ok in eq, otherwise kidney, cystitis(bladder), PLN
􏰂 purple - indicane (indol-sulphate) ok in eq, otherwise show bacterial overgrowth - catabolism
🔺 green – biliverdin ok in dog as they have enzyme, path in cat, others
🔺 brown - urobilinogen (UBG) ok in all animals, missing in posthepatic icterus!
􏰂 HNO3

Question 9

Gmelin result for healthy animal: exam Q

Answer 9

􏰁 condensed material on the surface of the glass tube - acidic urea (🐱)
🔺 yellow - urine itself
􏰁 white (opaque) – protein (🐴)
􏰁 purple - indicane (indol-sulphate)🐴
􏰁 green – biliverdin 🐶
🔺 brown - urobilinogen (UBG) 
🔺 HNO3

Question 10

Indocane

Answer 10

Indicane is a sulphated derivate of indol which is produced by bacteria in the lumen of the large intestines (colon and caecum) and it is absorbed to the blood and filtrated by the kidneys and finally excreted in the urine. When it is oxidised (i.e. by HNO3) it becomes purple. Appearance of indicane in the urine is normal in horses but in carnivores it indicates pathological enteral catabolism (enteritis, constipation).

Question 11

Ehrlich test

Answer 11

some drops of Ehrlich reagent in urine sample: checks UBG

Normal is mildly red from above and no color from the side

Abnormal is intensly red from above, mild/intense red from the side, and color change of the urine in general

Question 12

Prehepatic jaundice, how it works, three causes

Answer 12

Haemolytic crisis, free hgb, all br are incr in blood.

Hemolysis, Br ➡️ Liver ➡️ bile to feces and urine(portal vein)

🔺 babesiosis
🔺 immune hemolytic anemia
🔺 viper venom (snake)

Question 13

Hepatoc jaundice, disease, how it works

Answer 13

Liver damage - all br in blood are incr

Normal amount of hemolysis, Br ➡️ 20% taken up by liver, rest has to take extra rounds in blood(incr conc) ➡️ 60% is turned into BrII in liverm only 20% to bile duct, 40% back to blood(bile mix with blood!!)➡️ little BrII in feces, hypochromic. Since so much in blood ⬆️⬆️ but only some frome intestines ⬆️ UBG

Only 20% taken up - chirrosis! 80% dysfunction parenchyma

Question 14

Posthepatic jaundice, disease, how it works

Answer 14

Bile duct obstruction

Normal amount of hemolysis, Br ➡️ liver will transform to BrII, but then into blood bc duct obstruction so ⬆️⬆️⬆️ BrII but no UBG in blood!

🔺Pancreatitis is the most typical reason as it will squeese the bile duct 🔺Cholestasis: small/large, intra/extra hepatic duct obstruction - diplomatic term!

Question 15

🍏Lab result for prehepatic jaundice in blood

Answer 15

🔺Br I: ⬆️⬆️ hemolysis
🔺Br II: ⬆️ most stay in liver/bile
🔺UBG (not measured) ⬆️⬆️ intense colored feces
🔺Free haemoglobin (Hb) ↔️⬇️
🔺Haptoglobin cc. ⬇️
🔺Ht⬇️
🔺Reticulocyte count ⬆️⬆️⬆️ after some days, hemolysis!
🔺Coagulation parameters (PT, APTT, TT): ↔️⬆️ doesnt really change it, but if DIC present its bad!!
🔺Characteristic alterations in enzyme activities ⬆️ ALT/LDH: some from RBC
🔺Characteristic alterations in substrate concentrations: ⬆️ urea, liver catabolise globin to NH3 to urea

Question 15

🍏Lab result for hepatic jaundice in blood

Answer 15

🔺Br I: ⬆️ normal amount hemolysis, but extra rounds
🔺Br II: ⬆️⬆️ most go to blood not bile
🔺UBG (not measured) ⬆️pale feces
👎🔺Free haemoglobin (Hb) ↔️⬇️
🔺Haptoglobin cc. ↔️
👎 🔺Ht⬇️↔️
(👎 complicated disease - bile acids in blood!)
👎 🔺Reticulocyte count ↔️⬇️
🔺Coagulation parameters (PT, APTT, TT): ⬆️⬆️⬆️ vit K give no change bc no coag factors bc liver no work
🔺Characteristic alterations in enzyme activities ⬆️ ALT/AST/GLDH liver parench enzymes
🔺Characteristic alterations in substrate concentrations: ⬆️ NH3, ⬇️urea, ⬆️ bile accids

Question 16

🍏Lab result for posthepatic jaundice in blood

Answer 16

🔺Br I: ↔️ normal
🔺Br II: ⬆️⬆️⬆️ accumulation!
🔺UBG (not measured) - missing
🔺Free haemoglobin (Hb) ↔️
🔺Haptoglobin cc. ↔️
🔺Ht↔️
🔺Reticulocyte count ↔️
🔺Coagulation parameters (PT, APTT, TT): ⬆️⬆️ improves after vit K bc factors are present
🔺Characteristic alterations in enzyme activities ⬆️ ALKP, GGT bile obstruction enzymes
🔺Characteristic alterations in substrate concentrations: ⬆️ bile acids

Question 17

🍏Bilirubin in feces

Answer 17

Incr direct(2) excretion incr ubg and then stercobilin which have an orange color

Hyperchromic: intense color due to hemolysis - prehepatic jaundice

Hypocholic:
light brown: liver failure, less direct(2) br (hepatic jaundice)

light gray, acholic: bile duct obstruction -(posthepatic jaundice)

Question 18

🍏Urine parameters of Br, UBG and hgb in pre hepatic, hepatic, posthepatic jaundice

Answer 18

Prehep, hep, posthep:

Br: ⬆️, ⬆️⬆️, ⬆️⬆️⬆️ (brII)
UBG: ⬆️⬆️, ⬆️, ⬇️↔️ (missing)
Hgb: ⬆️⬆️⬆️, -, -

Question 20

Tests for liver ability to conjugate and then excrete

Answer 20

1️⃣Brom-sulphalein- (sulphobromtalein-) (BSP) retention test
2️⃣ Measurement of BSP half life
3️⃣ Indocyane green (ICG) retention test

Question 21

BSP

Answer 21

Brom-sulphalein retention test

• give function of UDP glucuronyl transferase activity:
• bromsulphalin IV admin - taken up by hepatocytes who splits it and conjugates with glucuronic acid - into bile

Question 22

Sampling procedure: BSP

Answer 22

1️⃣ Blood sampling for basal value (blank)
2️⃣ Administration of BSP iv (5 mg/kg body weight in 5% solution)
3️⃣ Blood sampling after 3 minutes for 100% value
4️⃣Blood sampling after 27 minutes (30 minutes after BSP administration) for the %-age in question.

Question 23

Result of BSP

Answer 23

Centrifuge and mix plasma with NaOH as BSP turns purple in alkaline environment! If its not conjugated it will be purple, spectrophotometry

• basal measurement is blank
• 3min sample as 100%

so liver dysfunction if more purple
- Retention of the BSP in plasma should be 5-10% in dog, 3-5% in cat (so around 95% is taken up by the liver)

Question 24

BSP halflife

Answer 24

Used in large animals bc its expensive. Measure every 5 mins until 45. Do it in a healthy animal first to make standard curve to use in sick animal measurement.

Question 25

ICG retention test

Answer 25

Indocyane green retention test: more expensive, less dangerous, good for cats

Blood sampling after 3 and 30mins

Normal is 20-25% retention in dog and 15%in cat

Question 26

Causes of incr BSP retention

Answer 26

Pene - primary liver failure:
- Liv - liver chorrosis
- Lever - liver tumor
- Hos - hepatic lipidosis
- Lasses - lipid mobilization syndrome
Dårlige - decr hepatic perfusion:
- Rakett - right sided heart failure
- På - PSS
- Aker - arteriole venous fistulas in liver
- Brygges - blockage in portal vessels
Osende - other causes
- Do - decr UDP-glucuronyl transferase activity in lover cells (conjenital)

Question 27

Incr bile acids in blood causes

Answer 27

LITEN - liver cell injury: bile acids into blood
BIL - bile duct obstruction: bile acids go to blood instead of bile
DURER - decr liver function: decr uptake or absorbed bile acids, incr ubg
PÅ - PSS

Question 28

Decr bile acids in blood (we say ba fail to increase not decrease.)

Answer 28

DRUE: decr absorption from intestines, intestinal wall damage or surgically removed ileum
SIL - severe liver cirrhosis, decr synth of bile acids

False negatives under these conditions!

Question 29

Measurement of bile acids, normal range

Answer 29

🔺 Spectrophotometric method: Na-EDTA or citrated blood!!
🔺 Sampling after starvation for 12h + after eating: 2 samples!
🔺measure total bile acids
🔺 20 μmol/L, 40 if not starved!

(For research: HPLC (High Performance Liquid Chromatography) - each bile acid is measured sep)

Question 30

When do we need bile acid measurement?

Answer 30

1️⃣dog looks ok but bad result
2️⃣ dog looks bad but ok result

Can be:
🔺Starved but still 40 μmol/L caused by lying owner or stress causing gall bladder constriction
🔺 16 μmol/L then feed 100 μmol/L can be PSS (can see CNS problems, small rbc)

Question 31

Role of bile acids

Answer 31

• detergent effect: smaller w/bigger surface fat droplets
• micelle formation
• lipid digestion
• neutralize gram- toxins

Question 32

Primary and secondary bile acids

Answer 32

Primary are prod in liver: cholic acid, chenodeoxycholic acid

Secondary are produced in the intestines: deoxycholic acid, lithocholic acid