Chemistry 2 > liver function 2 > Flashcards

liver function 2 Flashcards

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1
Q

bilirubin analysis

A

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

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2
Q

what is diazo reagent made up of

A

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

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3
Q

Evelyn Molloy Method Analysis

A
  • 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

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4
Q

Jendrassik- Grof Method of analysis

A

bilirubin+ diazo reagent = Azobilirubin + alkali ( blue color)

2 measurements are made on each sample :

  1. conjugated bilirubin ( without an accelerator )
  2. 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

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5
Q

calculating unconjugated bilirubin

A

unconjugated bilirubin = total bilirubin - conjugated bilirubin

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6
Q

Bilirubin analysis - sources of error

A
  • 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
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7
Q

method to measure different fractions of bilirubin

A

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)

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8
Q

bilirubin in neonates

A

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
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9
Q

increased bilirubin in neonates

A

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
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10
Q

method for neonatal analysis of bilirubin ( direct spectrophotometric method )

A

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

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11
Q

direct spectrophotometric method ( advantages & disadvantages )

A

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
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12
Q

Urine Bilirubin

A

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

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13
Q

Urine bilirubin methods of analysis

A

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

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14
Q

Urine bilirubin sample

A

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

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15
Q

clinical significance of Uirne bilirubin

A

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

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16
Q

Urine urobilinogen

A

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)

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17
Q

Urine urobilinogen : method of analysis

A

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

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18
Q

Urine urobilinogen specimen

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A

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
19
Q

Clinical Significant of Urine Urobinogen ( when increased & decreased)

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A

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)

20
Q

Fecal Urobilinogen

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A

examining the stool will detect low levels of urobilinogen
- clay-colored

semiquantitative methods are available & are the same as those used for urine urobilinogen

21
Q

expected urine bilirubin & urobilinogen in jaundice

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A

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
22
Q

Reference Ranges

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A

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

23
Q

Liver enzymes

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A

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

24
Q

Aminotransferases

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A
  • 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
25
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
26
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
27
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
28
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)
29
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
30
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
31
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
32
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
33
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
34
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)
35
Serum proteins- transferrin
- negative acute-phase reactant - major component of beta fraction on electrophoresis - function: transport iron deacreased in : - liver disease - infection - inflammation
36
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
37
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
38
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
39
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
40
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
41
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
42
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
43
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
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