Clinical Chemistry 2: Bilirubin Methods, Urobilinogen, and Hepatitis Infections

Vocabulary flashcards covering bilirubin methods (Malloy-Evelyn, Jendrassik-Grof), delta bilirubin, specimen handling, bilirubinometry, urobilinogen, Ehrlich reaction, and hepatitis infections (HEV, coinfection, superinfection, G, F).

Diazo reaction

Reaction of bilirubin with diazotized sulfanilic acid to form a colored product; the basis for most commonly used bilirubinometry methods.

Malloy and Evelyn

Developed the first clinically useful bilirubin quantitation method using classic diazo with 50% methanol as an accelerator.

Jendrassik and Grof

Described a diazo method using caffeine-benzoate-acetate as accelerator to solubilize bilirubin and measure total bilirubin.

Delta bilirubin

Conjugated bilirubin covalently bound to albumin; seen with significant hepatic obstruction; too large to be filtered by the glomerulus and excreted in urine.

Specimen: Serum

Serum used for bilirubinometry; fasting preferred and protected from light.

Specimen handling: stored in dark

Bilirubin tests should be protected from light and stored in the dark to prevent degradation.

Stability of samples

Bilirubin specimens are stable for: room temp 2 days, 4°C 1 week, and -20°C indefinitely.

Bilirubinometry scope

Primarily useful in neonates due to interference from carotenoids in adult serum.

Modification of Evelyn-Malloy Method

Modified approach that enables measurement of total bilirubin, conjugated bilirubin, and unconjugated bilirubin.

Total bilirubin

Sum of conjugated and unconjugated bilirubin measured by diazo-based methods.

Conjugated bilirubin (Direct)

Direct bilirubin; water-soluble form typically elevated in hepatobiliary obstruction.

Unconjugated bilirubin (Indirect)

Indirect bilirubin; not water-soluble; elevated in hemolysis or impaired conjugation.

Jendrassik-Grof principle

Bilirubin pigments react with diazo reagent (sulfanilic acid in HCl and nitrite) to form azobilirubin; two aliquots yield total and direct bilirubin.

Azobilirubin

Purple colored product formed by the diazo reaction; measured spectrophotometrically.

Wavelength for azobilirubin measurement

Absorbance typically read around 560 nm; reaction usually performed at pH 1.2.

Alkaline tartrate

Alkalinizes solution and shifts azobilirubin absorbance to a more intense blue color, enhancing measurement (often around 600 nm).

Ascorbic acid

Added to terminate the diazo reaction and destroy excess diazo reagent.

Methanol in Malloy-Evelyn method

Used as accelerator to solubilize unconjugated bilirubin.

Two aliquots in Jendrassik-Grof

One aliquot reacts with diazo reagent only; the second with diazo plus accelerator to yield total bilirubin.

Accelerator purpose

Accelerator solubilizes water-insoluble bilirubin fractions, yielding all fractions in serum.

Hemolysis and lipemia as error sources

Hemolysis and lipemia can interfere with bilirubin measurements and must be accounted for.

Ehrlich reagent

p-dimethylaminobenzaldehyde; reacts with urobilinogen to form a red color.

Urobilinogen (general)

Colorless end product of bilirubin metabolism; converted to urobilin in intestine and reabsorbed/excreted depending on liver function.

Ehrlich units (urine urobilinogen)

Units used to express urobilinogen concentration in urine (normal ranges provided in standard tables).

Ascorbic acid in urobilinogen assay

Reducing agent that keeps urobilinogen in reduced state during analysis.

Saturated sodium citrate in Ehrlich reaction

Stops reaction and minimizes formation of chromogens other than urobilinogen.

Urine urobilinogen specimen

Fresh 2-hour urine used; readings should be done promptly; reported in Ehrlich units.

Fecal urobilinogen

Semiquantitative test using aqueous extract of fresh feces; alkaline ferrous hydroxide reduces urobilinogen and allows semiquantitation.

Hepatitis E (HEV)

Virus causing hepatitis; zoonotic in industrialized countries and waterborne in developing countries; fecal-oral route; incubation 21–42 days; usually mild but severe in pregnancy; diagnosed by IgM anti-HEV or rising IgG anti-HEV.

Coinfection (HBV)

HBV infection occurring concurrently with another hepatitis virus; may impact chronicity.

Superinfection (HBV)

A new hepatitis virus infects someone with established HBV infection; may lead to severe hepatitis; chronicity risk similar to HBV infection alone.

Hepatitis G

Form of hepatitis with unclear role in disease.

Hepatitis F

Enteric agent that may be transmitted to primates.