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
Urine urobilinogen specimen
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
Clinical Significant of Urine Urobinogen ( when increased & decreased)
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)
Fecal Urobilinogen
examining the stool will detect low levels of urobilinogen
- clay-colored
semiquantitative methods are available & are the same as those used for urine urobilinogen
expected urine bilirubin & urobilinogen in jaundice
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
Reference Ranges
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
Liver enzymes
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
Aminotransferases
- 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
Phosphatases ( ALP)
- ALP
- most useful for differentiating hepatobiliary disease from osteogenic bone disease ( liver & bone)
- highest levels found in extrahepatic obstructions
- can be found in absence of liver disease aswell so test with other tests as well
Phosphotases ( 5’- nucleotidase)
5’- nuclotidase ( 5NT)
significant increases in hepatobiliary disease
not found in the bone, so can be used to differentiate increases in ALP
in liver disease:
- inceased ALP
- increased 5NT
in bone disease:
- increased ALP
- N or sl increased 5NT
gamma- Glutamylertransferase ( GGT )
- plays a role in differentiating the cause of elevated ALP concentrations ( similar to 5NT)
- highest levels seen in biliary obstruction
- GGT will rise with ingestion of alcohol or certain drugs
Lactate dehydrogenase (LD)
- widely distributed throughout the body
- high levels found in metastatic carcinoma of the liver
- moderate elevations seen in viral hepatitis & cirrhosis
- slight elevations in biliary tract disease
- fractionation of LD into its isoenzymes can provide useful information regarding the site of origin when there is a rise in LD ( 4&5)
Serum Proteins
useful to assess synthetic ability of the liver
- most serum proteins synthesized by the liver exceot immunoglobulins
chronic liver disease
- decreased albumin ( also in acute liver disease but to a less extent )
- decreased alpha- globulin
- increased gamma-globulin
serum protein - Albumin
protein present in the highest concentration in plasma
functions
- responsible for 80% of colloid osmotic pressure of the intravascular fluid ( maintains fluid balance in tissue)
- negative acute-phase reactant ( decreases during inflammation)
- transport of various substances ( binds to unconjugated bilirubin )
Hypoalbunemia in
- Cirrohsis
- Autoimmune hepatitis (AIH)
- Alcoholic hepatitis
Serum proteins - Transthyretin
short half life of 24-48 hrs
sensitive marker of current synthetic ability
transport protein for: vitamin A & thyroxine (T4)
commonly used as a measurement of nutritional status
Serum protein - Ceruloplasmin
copper- containing glycoprotein synthesized in the liver
positive acute phase reactant
90% of serum copper is bound to cerulplasmin ( remaining 10 % bound to albumin)
decreased in :
- WIlson’s disease
- Cirrhosis
- Chronic hepatitis
increased in:
- inflammation
- Cholestatis
- Hemochromatosis
- Pregnancy
Serum protein - alpha 1- antitrypsin
major serine protease inhibitor (serpin) in plasma
positive acute phase reactant
decreased in :
- alpha 1- antitrypsin
- Cirrhosis
increased in :
- acute inflammation
Serum protein - alpha fetoprotein ( AFP)
- synthesized in utero by developing embryo & then by parenchymal cells in the liver
- levels fall to adult values by 1 year of age
increased in:
- acute & chronic hepatitis ( mild increase)
- hepatocellular carcinoma (HCC)
Serum proteins- transferrin
- negative acute-phase reactant
- major component of beta fraction on electrophoresis
- function: transport iron
deacreased in :
- liver disease
- infection
- inflammation
serum proteins - complement
complement C3 is most abundant, followed by C4
function - natural defense - protects from infections
decreased in
- autoimmun disease
- chronic hepatitis
increased in
- inflammatory disease
Serum proteins - C- Reactive Protein ( CRP)
general indicator of inflammation
positive acute- phase reactant
elevated levels are associated with increased risk for coronary heart disease & stroke
increased in:
- acute inflammation
- Myocardial infarctions
- Bacterial or viral infections
Coagulation Proteins
coagulation proteins & inhibitors of the coagulation system are synthesized in the liver
Synthesized by hepatocytes :
- fibrinogen
- prothrombin
- factor V, VII, IX, X, XII
- protein C & S
- Antithrombin
Synthesized by sinusoidal endothelial cells :
- Factor VII
- von Willebrand factor
Coagulation proteins - fibrinogen
- one of the largest proteins in plasma
- positive acute- phase reactant
- function: form fibrin clots when activated by thrombin
decreased in:
- extensive bleeding
- liver disease
increased in:
- inflammatory processes
- pregnancy
Prothrombin Time (PT/INR)
increased in liver disease
- liver is unable to manufacture enough clotting factor
- or disruption in bile flow leads to inadequate absorption of vitamin K from intestine
not routinely used to help diagnose liver disease
serial measurements can help monitor the progression of disease & the risk of bleeding
very prolonged PTs indicate severe liver disease & a poor prognosis
Urea
synthesized in the liver from amino groups ( -NH2) & free ammonia from protein catabolism
- catalyzed by enzymes in the urea cycle
low levels of urea found in the end-stage liver disease
reference range
serum/plasma: 2.1-7.1 mmol/L
protein - amino acids - ammonia - urea
Ammonia
the liver converts ammonia to urea so that it can be removed by the kidneys
- ammonia levels reflect the liver’s ability to perform this conversion
in liver failure:
- increased ammonia
- hepatic coma
patients become disoriented & lapse into unconsciousness
ammonia levels >200µmol/L are associated with cerebral edema & poor prognosis in acute liver failure patients ( good indication of liver failure)
reference range : 11-35µmol/L
Serological Markers for hepatitis infections
hepatitis A ( aka infectious hepatitis)
- IgM anti-HAV
- IgG anti-HAV
hepatitis B ( aka serum hepatitis or long-incubation hepatitis )
- HBcAg- antigen in core of virus
- HBsAg- antigen on surface protein
- HBeAg
- IgM anti-HBc
- Anti-HBs
hepatitis C
- Anti-HCV
