liver function 2 Flashcards
bilirubin analysis
methods are based on Van den Berg reaction
- reaction of bilirubin with a diazo reagent ( with or without an accelerator ) to form azobilirubin
2 Most common methods:
- evelyn-molloy method ( older)
- Jendrassik-Grof Method ( more common)
bilirubin + diazo = Azobilirubin
what is diazo reagent made up of
dulfanilic acid in HCl + sodium notrite ( diazorized sulfanilic acid)
Evelyn Molloy Method Analysis
- done at an acid pH of 1.2
- Methanol is the accelerator ( Molloy & Methanol)
- 2 measurements are taken
1. total bilirubin ( with an accelerator)
2. conjugated bilirubin ( without an accelerator)
unconjuagted bilirubin = total bilirubin - conjugated bilirubin
Jendrassik- Grof Method of analysis
bilirubin+ diazo reagent = Azobilirubin + alkali ( blue color)
2 measurements are made on each sample :
- conjugated bilirubin ( without an accelerator )
- total bilirubin ( with an accelerator)
accelerator used : caffeine sodium benzoate
after a period of time ascorbic acid is added to destroy excess diazo reagent & stop reaction
alkaline tartrate is added to make pH alkaline ( & get blue color)
measured at 600nm
the intensity of the blue color is directly proportional to the amount of bilirubin in the sample
calculating unconjugated bilirubin
unconjugated bilirubin = total bilirubin - conjugated bilirubin
Bilirubin analysis - sources of error
- bilirubin standard should be carefully prepared as they will deteriorate when exposed to light
- hemolysis & lipemia will alter bilirubin concentrations
- hemoglobin will compete with diazo reagent & give falsely low results
- specimens should be stored refrigerated & in the dark until testin is performed
method to measure different fractions of bilirubin
bilirubin + diazotized sulfanic acid + accelerator = 2 azobilirubin ( total bilirubin)
bilirubin + diazotized sulfanic acid = 2 azobilirubin ( conjugated bilirubin )
total bilirubin - conjugated bilirubin = unconjugated bilirubin ( indirect bilirubin)
bilirubin in neonates
neonates = children < 1 month of age
specimen collectde : capillary sample from finger or heal ( avoid hemolysis )
before birth, unconjugated bilirubin is cleared by the placenta
the enzyme UDP- glucuronyl transferase is needed to conjugated bilirubin
- one of the last enzymes to develop in newborns
- results in increased unconjugated bilirubin in the first weeks of life
increased bilirubin in neonates
causes jaundice - yellowing of the skin
yellow sclera ( white of eyes)
Kernicterus- brain damage
- the blood-brain barrier is not mature in neonates; therefore, lipid soluble unconjugated bilirubin can get through
- bilirubin deposits on brain which can be fatal
method for neonatal analysis of bilirubin ( direct spectrophotometric method )
principle
- absorbance of bilirubin at 454 nm is proportional to its concentration
- problem: oxyhemoglolobin ( HbO2) also absorbs at this wavelength
- a 2nd reading is taken at 540 nm
- oxyHb absorbs at both wavelengths, but bilirubin doesn’t
- absorbance due to bilirbin = A454 - A540
this technology is built into bilirubinometers
direct spectrophotometric method ( advantages & disadvantages )
advantages
- fast TAT
disadvantages
- only total bilirubin can be measured
- only useful for infants
- older children & adults have pigment that will react (eg. carotene) & falsely increase results
Urine Bilirubin
normal urine is bilirubin NEGATIVE
bc unconjugated bilirubin isn’t water soluble & cannot be filtered out by the kidneys
if urine bilirubin is positive it will be due to an increase in conjugated bilirubin
urine with increased bilirubin with be dark yellow
Urine bilirubin methods of analysis
dipstick - most common
- test pad contains diazo reagent
- dip test strip in urine & wait 30 secs
- compare color of test strip to the color chart
- neg, 1+, 2+, 3+
Tablet test - ictotest
- mores sensitive ( detects smaller amounts)
- add 10 drops of urine to mat ( bilirubin will absorb on mat)
- add tablet & 2 drop of water
- blue color = positive for bilirubin
- not often used now
both methods measure conjugated bilirubin bc only conjugated bilirubin is found in urine ; unconjugated bilirubin isnt water soluble
Urine bilirubin sample
both methods need fresh sample; bilirubin will disappear on standing
bilirubin diglucuronide ( conjugated bilirubin) can be :
- hydrolyzed to free bilirubin ( less reactive) OR
- oxidized to biliverdin ( nonreactive )
Protect sample from light
- bilirubin is photosensitive
- up to 50% decrease/hour due to light
refrigerate for 24hrs max
clinical significance of Uirne bilirubin
urine bilirubin is increased in :
- hepatitis
- cirrhosis
- other liver disorders
- bilirary obstruction ( gallstones, carcinoma)
large amounts of bilirubin in the urine can be present in hepatic disorders
in some cases bilirubin crystals will be see under microscopic examination
Urine urobilinogen
urobilinogen
- consists of urobilinogen, mesobilinogen, stercobilinogen
- colorless reduction products of bilirubin
- up tp 20 % enters portal circulation & goes to the liver
- on the way to the liver, 2-5 % enter general circulation & get filtered by kidneys
it is normal to have a small amount of urobilinogen in the urine ( 0.2-1 mg/dL)
Urine urobilinogen : method of analysis
principle:
Ehrlich’s reagent + urobilinogen = red product
Erlich’s regaent = p-dimethylaminobenzaldehyde ( red product)
OR
Diazonium salt + urobilinogen = red azo dye
an be spectrophotometric or dip stick
note: false neg results may occur with old specimens
