liver function 2

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

dulfanilic acid in HCl + sodium notrite ( diazorized sulfanilic acid)

• 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

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

unconjugated bilirubin = total bilirubin - conjugated bilirubin

• 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

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)

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

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

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

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

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

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

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

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

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)

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

timed specimen is usually best • usually a 2 hr collection between 1-3 or 2-4 pm • this is the time of maximum excretion of urobilinogens urobilinogen is unstable • protect from light • keep cool • analyze immediately a 24hr collection can be used • collect in a dark bottle • add 100 mL of toluene- protect urine from air & prevents bacterial growth

early detection of liver disease urine urobilinogen is increased in: • liver disorders } when damaged, the liver cannot extract • hepatitis } the reabsorbed urobilinogen from the portal circulation, so it is • cirrhosis } filtered by the kidneys & excreted in the urine • hemolytic disorders urine urobilinogen is decreased : - partial or complete obstructions ( post-hepatic obstructions)

examining the stool will detect low levels of urobilinogen - clay-colored semiquantitative methods are available & are the same as those used for urine urobilinogen

hemolytic disease • urine bilirubin negative • urine urobilinogen +++ • type of jaundice: pre hepatic liver damage • urine bilirubin + or - • urine urobilinogen ++ • type of jaundice : hepatic Bile Duct Obstruction • urine bilirubin +++ • urine urobilinogen normal or - • type of jaundice: post hepatic

serum/plasma • conjugated bilirubin: 0-3µmol/L • unconjugated bilirubin: 3-14 µmol/L • total bilirubin: 3-17 µmol/L urine • urine bilirubin: negative • urobilinogen: 0.1-1.0 Ehrlich units / 2 hrs 0. 5-4.0 Ehrlich units / 24hrs Ehrlich units =1 mg urobilinogen

play an important role in the assessment of liver function - enzymes are released into circulation when there is an injury to the liver most clinically useful enzymes: • ALT • AST • ALP • 5’-nucleotidase • GGT • LD useful for differentiating between heparocellular ( functional ) & obstructive (mechanical) liver disease

• AST & ALT • Most useful for detecting hepatocellular damage to the liver • ALT is mote liver specific than AST ( most found in the liver with smaller amounts in the kidney & skeletal muscle) highest levels are found in acute conditions • viral heaptitis* • drug & toxin - induced liver necrosis • hepatic ischemia