Sect 5.2 - Test Methods & Liver Enzymes

water solubility

Bilirubin measurement is based on:

Conjugated (direct) and Albumin-bound (delta)

water soluble -- directly react with substances

Unconjugated (indirect)

insoluble in water -- an accelerator must be used to make it soluble; then can be measured directly

Jendrassik-Grof method

Evelyn-Malloy method

HPLC (High performance liquid chromatography)

3 Bilirubin measurement methods:

HPLC (High performance liquid chromatography)

Which method is considered the "gold standard"?

Jendrassik-Grof method

Which method is most common & preffered?

also most sensitive

Caffeine Sodium Benzoate (Jendrassik-Grof method)

Duponol (Evelyn-Malloy method)

Identify substance that is used to convert all forms of bilirubin to a soluble form:

aka the accelerators

Total - Conjugated = Unconjugated

Calculation for total and conjugated bilirubin to determine the unconjugated amount

Jendrassik-Grof method

Uses 2 samples: Conj and Total

- Direct (Conj) serum reacts with diazotized sulphanilic acid to form red colored compound

- Total: pretreated with caffeine sodium benzoate (accelerator), then reacts with diazotized sulphanilic acid

Conversion of red acid bilirubin → blue-green alkaline bilirubin

Shift the pH to alkaline (pH 13)

Change in color (red →blue-green)

Jendrassik-Grof method

Solutions of diazo rgt, ascorbic acid, alkalkine tartrate, and dilute HCl does what to the product?

pH of 13 (alkaline) and blue-green product

Jendrassik-Grof method occurs at what pH and what color product is measured?

less subjected to interfering chromagens

Jendrassik-Grof method

Advantage of blue-green alkaline bilirubin

Evelyn-Malloy method

Uses 2 samples: Conj and Total

- Direct (Conj) serum + sulfanilic acid reacts with sodium nitrate to form diazotized sulfanilic acid

- Total: In the presence of Duponol (accelerator), total bilirubin reacts with diazotized sulfanilic acid to form azobilirubin

pH of 1.3 (acidic) and red-colored product

Evelyn-Malloy method occurs at what pH and what color product is measured?

serum

What is the preferred specimen for Bilirubin measurement?

**can also use: plasma, spinal fluid, urine

interferences with Bilirubin Measurement

False increase: hemolysis, lipemia (add color)

False decrease: UV light or heat (breaks bilirubin down)

conjugated

What type of bilirubin is excreted in the urine?

Ictotest

Urine bilirubin confirmatory method

- mores specific/sensitive

- pos = purple

true

T or F:

Always go with the result of the Ictotest (even if dipstick gives a different result)

Spectrophotometric measurement

Neonatal Bilirubin measurement utilizes:

measure oxyhemoglobin and bilirubin

What two measurements are taken in the spectrophotometric analysis for neonatal bilirubin measurement?

total bilirubin (subtract oxyhemoglobin and bilirubun)

What is being measured in the spectrophotometric analysis for neonatal bilirubin measurement?

hemolysis and lipemia have no effect

Does hemolysis and lipemia have an effect on the spectrophotometric analysis for neonatal bilirubin measurement?

newborns have a controlled diet of breast milk/formula

Dietary pigments can affect specimen used in spectrophotometric analysis

Why doesn't it cause a problem with newborn specimens?

P-dimethylaminobenzaldehyde

What is the Ehrlich's reagent

Urobilinogen

Urine/fecal specimen + alkaline ferrous hydroxide → urobilinogen + Erlich's reagent → sodium acetate → forms red color

alkaline ferrous hydroxide

enzyme that converts urobilin/stercobilin to urobilinogen

Sodium acetate

enzyme used in urobilinogen measurement that reduces interference form other chromogens

interferences with Urobilinogen

False increase: Porphobilinogen (red color), Bilirubin (amber color)

bilirubin can be precipitated out with barium chloride & removed by filtration

enzymes

released from cells when they are diseased

true

T or F:

Increase in enzymes are elevated before S/S of disease show

1. release from cells due to cellular damage

2. changes in enzyme production rate as a result of genetic/malignancy problems

Enzyme levels reflect (2):

AST

ALT

GGT

ALP

LDH

Liver enzymes (5):

equal amounts in heart, skeletal muscle, and liver

What is the tissue source of AST?

predominantly found in the liver

What is the tissue source of ALT?

primarily in liver and bile ducts

also in pancreas, kidney, spleen, intestine

What is the tissue source of GGT?

primarily liver and bone, also placenta, kidney, intestine

What is the tissue source of ALP?

found in equal amounts all over the body

What is the tissue source of LDH?

ALT

What is the most specific liver enzyme?

AST

enzymes: AST and malate dehydrogenase (MDH)

product measured: decreased abs of NADH

ALT

enzymes: ALT and lactate dehydrogenase (LD)

product measured: NAD+

2:1

Normal AST:ALT ratio

massive cell necrosis

AST higher than ALT means

(hepatocytes produce ALT, decrease ALT = decrease hepatocytes)

acute liver disease

ALT higher than AST means

(hard to differentiate liver disease based on ALT and AST alone)

Wilson's Disease

AST:ALT ratio of >4

Copper accumulation in liver (No protein to carry Cu)

GGT

enzymes: GGT

product measured: p-nitroaniline

liver

Majority of GGT serum activity is from:

early hepatobiliary disease

GGT is one of the most sensitive tests for detection of:

90% reliability; but not specific for the liver

bone and liver disease

GGT is often used in correlation with ALP to differentiate between:

bone disease

increase ALP: decreased GGT

liver diease

decreased ALP: increased GGT

ALP

enzymes: ALP

product: 4-ntrophenoxide (yellow product)

increased synthesis, not release from damaged/necrotic cells

Increases seen with ALP is associated with:

interferences to enzymatic measurement methods?

Temperature needs to be maintained at 37 C for accurate product formation

Free from hemolysis (many measure NADH which is contained in red cells)

Alcohol and drugs

Loss of stability over time (test right away or fridge for 3 hours or freezer for 30 days)

need to maintain substrate concentration in excess is important to keep reaction moving in zero order

GGT and ALP testing is measured using kinetic reactions, what extra step must we take?

interferences with ALP testing

less stable; specimen must be < 3 hours old

anticoagulants that remove calcium and magnesium will prevent product formation

Isoenzymes

forms of the same enzyme that arise from unique gene sequences

Zone electrophoresis

Most common method of measuring isoenzymes

can also use: Ion-exchange chromatography, Selective inactivation, Immunoassay

Cathode (-)

Anode (+)

In any device which consumes power:

Cathode (+)

Anode (-)

In any device which produces power:

Cathode

Where is the sample application point in electrophoresis?

Cathode (-) to Anode (+)

What is the migration direction in electrophoresis?

Placental

Slowest, greatest net + charge

Rare/Artifact/Other

fastest, closest to + anode, greatest net - charge

placental (slowest, greatest net + charge)

intestine

bone

liver

fast liver (obstruction)

rare(ectopic)/artifact(debris) (fastest, closest to + anode)

List the mobility pattern of ALP isoenzymes from the point of application (cathode)

LD-5

LD isoenzyme correlates best with metastatic liver disease

5-NT & LAP

both enzymes used to increase specificity of ALP enzyme for liver involvement

both enzymes usually correspond to ALP concentrations (give essentially same information concerning obstructive disease but do not increase with bone involvement)

LAP

usually associated with pancreatic carcinoma when there is also biliary involvement

5-NT

usually associated with biliary obstruction

used to tell if the high ALP is from bone or liver origin -- will not be increased in bone involvement

Why might a physician order a 5'-NT and LAP?

acute viral hepatitis

high AST/ALT (>1000ul) level indicates:

alcoholic cirrhosis

high GGT level indicates:

ALP, GGT, 5'-NT

What enzyme levels are elevated in obstructive disorders?

GGT

What enzyme level is elevated in hepatobiliary disease?

ALP, LD, GGT, 5'-NT

What enzyme levels are elevated in metastatic liver disease?

growing activity

pregnancy (found in placenta(

When might an elevated ALP be normal?

long-term condition, so enzyme levels will not be increased long-term

Why is there not much enzyme activity in chronic liver disease and cirrhosis?