ASSESSMENT OF LIVER FUNCTION/LIVER

FUNCTION TESTS

Blirubin
-The reaction of bilirubin with a diazotized sulfanilic acid
solution to form a colored product.
-Ehrlich in 1883 using urine samples
-Van den Bergh found that the
diazo reaction may be applied to serum samples but only
in the presence of an accelerator (solubilizer).
-Malloy and Evelyn developed the first
clinically useful methodology for the quantitation of
bilirubin in serum samples using the classic diazo reaction with a 50% methanol solution as an accelerator.
In
1938, Jendrassik and Grof described a method using the
diazo reaction with caffeine-benzoate-acetate as an accelerator.
-Today, all commonly used methods for measuring bilirubin and its fractions are modifications of the
technique described by Malloy and Evelyn.

Total bilirubin and conjugated bilirubin - measured

Unconjugated bilirubin = TB – B2

Bilirubinometry – only useful in the neonatal population because of the presence

of carotinoid compounds in adult serum that causes
strong positive interference in the adult population.
 measurement of reflected
light from the skin using two wavelengths that provide a
numerical index based on spectral reflectance; new generation uses microspectro.
 Determines the optical densities of bilirubin,
hemoglobin, and melanin in the subcutaneous layers of
the infant’s skin.

Unconjugated (indirect) bilirubin is a nonpolar and

water-insoluble substance that is found in plasma bound to albumin; will only react with the diazotized sulfanilic
acid solution (diazo reagent) in the presence of an accelerator (solubilizer).
Conjugated (direct) bilirubin is
a polar and water-soluble compound that is found in
plasma in the free state (not bound to any protein). This type of bilirubin will react with the diazotized sulfanilic
acid solution directly (without an accelerator).
Delta bilirubin, when present, will react
in most laboratory methods as conjugated bilirubin.
Thus, total bilirubin is made up of three fractions: conjugated, unconjugated, and delta bilirubin.

SPECIMEN COLLECTION AND STORAGE

-Total bilirubin methods using a diazotized sulfanilic acid

solution may be performed on either serum (preffered by Malloy-Evelyn because of alcohol’s absence) or plasma.
-A fasting sample is preferred as the presence of lipemia will increase measured bilirubin concentrations.
Hemolyzed samples should be avoided as they may decrease the reaction of bilirubin with the diazo reagent.
-Specimens should be protected from light. Or else values may reduce
by 30%–50% per hour. If serum or plasma is separated from the cells and stored in the dark, it is stable for
 2 days at room temperature
 1 week at 4°C
 indefinitely at 20°C.
American Association for Clinical Chemistry and the National Bureau of Standards candidate reference method
for total bilirubin = modified Jendrassik-Grof procedure using caffeine-benzoate as a solubilizer.

Jendrassik Grof or Malloy-Evelyn procedure - most frequently

used method to measure bilirubin.
Jendrassik-Grof method is slightly more complex, but it has the following
advantages over the Malloy-Evelyn method:

■Not affected by pH changes

■Insensitive to a 50-fold variation in protein concentration of the sample
■Maintains optical sensitivity even at low bilirubin
concentrations
■Has minimal turbidity and a relatively constant serum
blank
■Is not affected by hemoglobin up to 750 mg/dL

Malloy-Evelyn Procedure
-Bilirubin pigments in serum or plasma are reacted with a
diazo reagent. The diazotized sulfanilic acid reacts at the
central methylene carbon of bilirubin to split the molecule forming two molecules of azobilirubin.
-pH 1.2
-End color: red-purple
-560 nm absorption
-most commonly used accelerator: methanol

Jendrassik-Grof Method for Total and Conjugated

Bilirubin Determination

Principle: Bilirubin pigments in serum or plasma are reacted with a diazo reagent (sulfanilic acid in hydrochloric
acid and
sodium nitrite), resulting in the production of the purple product azobilirubin.
-Solubilizer: Caffeine-benzoate
-The reaction without the accelerator will yield
conjugated bilirubin only.
. The ascorbic acid destroys the excess diazo reagent.
-Alkalinized using an alkaline tartrate solution (which makes the azobilirubin a more intense blue)
-End color: blue
-At 600 nm
-The intensity of color produced directly proportional to bilirubin concentration.
Urobilinogen in Urine and Feces

-Urobilinogen is a colorless end product of bilirubin metabolism that is oxidized by intestinal bacteria to the
brown pigment urobilin.
-In the normal individual, part of the urobilinogen is excreted in feces
-The remainder is reabsorbed into the portal blood and returned to the liver.
-A small portion that is not taken up by the hepatocytes is excreted by the kidney as urobilinogen.
-Increased levels of urinary urobilinogen are found in
 hemolytic disease
 defective liver-cell function (hepatitis)
-Absence of urobilinogen from the urine and stool is
 most often seen with complete biliary obstruction
-Decreased in
 biliary obstruction
 HCC
-quantitative methods for urobilinogen are based on a reaction first described by Ehrlich in 1901
-Semiquantitative (urine) p-dimethyl aminobenzaldehyde (Ehrlich’s reagent) = red color.
-Ascorbic acid as a reducing agent to maintain urobilinogen in the reduced state.
-Saturated sodium acetate stops the reaction and minimizes the combination of other chromogens with the
Ehrlich’s reagent.
Specimen: A fresh 2-hour urine specimen (to prevent oxidation of urobilinogen to urobilin) is collected. This
specimen should be kept cool and protected from light. Spectrophotometric readings should be made within 5
minutes after color
production because the urobilinogen-aldehyde color slowly decreases in intensity.

-Compounds that also react with Ehrlich’s reagent

  porphobilinogen,
 sulfonamides,
 procaine
 5-hydroxyindoleacetic acid.

-Bilirubin will form a green color and, therefore, must be removed, as previously described
-Reference Range
 0.1–1.0 Ehrlich units every 2 hours or
 0.5–4.0 Ehrlich units per day (0.86.8 mmol/day);
1 Ehrlich unit is equivalent to approximately 1 mg of urobilinogen.

Fecal Urobilinogen
-Same principle
-Urobilin present is reduced to urobilinogen by treatment with alkaline ferrous hydroxide
before Ehrlich’s reagent is added.
Reference range:
 75–275 Ehrlich units per 100 g of fresh feces or
 75–400 Ehrlich units per 24-hour specimen

Serum bile acid

-levels are elevated in liver disease, the total concentration is extremely variable and adds no diagnostic
value to other tests of liver function.
-rarely performed because the methods required are very complex
-More relevant information of liver dysfunction may be gained by examining patterns of individual bile acids and
their state of conjugation.

Enzymes
-Injury to the liver resulting cytolysis or necrosis will cause the release of enzymes into circulation.
-Play an important role in differentiating hepatocellular (functional) from obstructive (mechanical) liver
disease

Aminotransferases
-AST (formerly referred to as serum glutamic-oxaloacetic transaminase [SGOT])
ALT (formerly referred to as serum glutamic-pyruvic transaminase [SGPT]).
-The aminotranferases are for catalyzing the conversion of aspartate and alanine to oxaloacetate and pyruvate,
respectively.
In the absence of acute necrosis or ischemia of other organs, these enzymes are most useful in the detection of
hepatocellular (functional) damage to the liver.
 ALT is found mainly in the liver (lesser amounts in skeletal muscle and kidney);
 more “liver-specific; increase in ALT activity is
usually greater than that for AST
 AST is widely distributed in equal amounts in the heart, skeletal muscle, and liver,

-Regardless, the serum activity of both transaminases rises rapidly in almost all diseases of the liver
-Remain elevated for up to 2–6 weeks.
-Highest levels (found in acute conditions) such as
 viral hepatitis
 drug- and toxin-induced liver necrosis
 hepatic ischemia.
-Mildly elevated in obstructive liver damage.
-Elevations in these enzymes may be a result of other organ dysfunction or failure
 acute myocardial infarction
 renal infarction
 progressive muscular dystrophy,
 secondary liver disease such as
o infectious mononucleosis
o diabetic ketoacidosis,
o hyperthyroidism.
-Decrease in
 severe acute hepatitis (owing to the
exhaustive release of hepatocellular enzymes)

Phosphatases
Alkaline Phosphatase
-zinc metalloenzymes; widely distributed in all tissues;
-However, highest activity is seen in the
 liver
 bone,
 intestine
  kidney, and
 placenta.
-clinical utility of ALP lies in its ability to differentiate hepatobiliary disease from osteogenic bone
disease.
-In the liver, the enzyme is localized to the microvilli of the bile canaliculi (great marker of extrahepatic biliary
obstruction)
-Very high concentrations: extrahepatic obstruction
- Slight to moderate increase : hepatocellular disorders such as hepatitis and cirrhosis.
-Others that will elevate ALP
 Paget’s disease, bony metastases, diseases associated with an increase in osteoblastic activity,
and rapid bone growth during puberty. (bone-related)
 In pregnancy (may remain elevated up to several weeks post delivery)
5-Nucleotidase
-For catalyzing the hydrolysis of neucleoside-5-phosphate esters.
-Elevated in hepatobiliary disease
-Useful in differentiating ALP elevations due to the liver from other conditions where ALP may be seen in
increased concentrations (bone diseases, pregnancy, and childhood growth).
Levels of both 5NT and ALP are elevated in liver disease; in primary bone disease, ALP level is elevated, but
the 5NT level is usually normal or only slightly elevated.
-Much more sensitive to metastatic liver disease than is ALP because, unlike ALP, its level is not significantly
elevated in other conditions
-Some increase in enzyme activity may be noted after abdominal surgery

-Glutamyltransferase
-Membrane-localized enzyme ; hepatic microsomal enzyme
-High concentrations in the
 Kidney
 liver
 pancreas
 intestine, and
 prostate
-Plays a role in differentiating the cause of elevated levels of ALP as the highest levels of
GGT are seen in biliary obstruction.
-Elevates because of ingestion of
 alcohol
 certain drugs (barbiturates, tricyclic antidepressants, and
anticonvulsants)
-Sensitive test for cholestasis caused by chronic alcohol or drug ingestion.
-Measurement of this enzyme is also useful if jaundice is
absent for the confirmation of hepatic neoplasms.

Lactate Dehydrogenase
-With a very wide distribution throughout the body. Non-specific marker of cellular injury.
-Released into circulation when cells of the body are damaged or destroyed.
-Elevations of total serum LDH levels are common in
 acute viral hepatitis (moderate)
 cirrhosis (moderate)
 biliary tract disease (slight)
 metastatic carcinoma of the liver (high)
-Fractionation of LDH into its five tissue-specific isoenzymes may give useful information about the site of origin
of the LDH elevation.

TESTS MEASURING HEPATIC SYNTHETIC ABILITY

-Decreased serum albumin = decreased liver protein synthesis, found more often in chronic.
-a-globulins decrease = chronic liver disease.
-Low/absent a-globulin = a-antitrypsin deficiency as the cause of the chronic liver disease.
-Serum y-globulin transiently increased = acute liver disease; and remain elevated in chronic liver disease.
-The highest elevations
 chronic active hepatitis and
 postnecrotic cirrhosis.
-IgG and IgM levels are more consistently elevated in chronic active hepatitis;
-IgM, in primary biliary cirrhosis;
-IgA, in alcoholic cirrhosis

Prothrombin time - commonly increased in liver disease (because the liver is unable to manufacture adequate
amounts of clotting factor or because of inadequate absorption of vitamin K from the intestine).
-not routinely used to aid in the diagnosis of liver disease.
-Only in following the progression of disease and the assessment of the risk of bleeding.
-A marked indicates severe diffuse liver disease and a poor prognosis.

Tests Measuring Nitrogen Metabolism

-The liver plays a major role in removing ammonia from the bloodstream and converting it to urea so that it can
be removed by the kidneys.
-A plasma ammonia level is a reflection of the liver’s ability to perform this conversion.
-In liver failure, ammonia and other toxins increase in the bloodstream and may ultimately cause
hepatic coma (cause is not fully known).
 patient becomes increasingly disoriented
 gradually lapses into unconsciousness.
-Ammonia levels are most useful when multiple measurements are made over time.
The most common laboratory determination of ammonia concentrations is based on the following reaction:

-NADP+ formed is measured at 340 nm.

-SOC: plasma collected in EDTA, heparin, or potassium oxalate
-Samples should be immediately placed on ice to prevent metabolism of other nitrogenous compounds to
ammonia.
-Frozen samples are stable for several days.
Hemolyzed samples should be rejected -- as red blood cells have a concentration of ammonia 2–3 times higher
than that of plasma

Hepatitis
-Injury to the liver characterized by presence of inflammation in the liver tissue.
Viral infections account for the majority of hepatitis cases observed in the clinical setting (but can also be caused
by other infectious and non-infectious).
-SYMPTOMS:
 jaundice
 dark urine
 fatigue
 nausea
 vomiting
 abdominal pain.
Hepatitis A
-Also known as infectious hepatitis or short-incubation hepatitis, is the most common form worldwide
-Caused by a nonenveloped RNA virus of the Picornavirus family.
-Excreted in bile and shed in the feces, which can contain up to 10^9 infectious virions per gram
-Symptoms are generally self-limited and resolve within 3 weeks.
However, in rare instances, patients develop fulminant liver failure.
-Immunoglobulin M (IgM) antibodies to HAV (IgM anti-HAV) are detectable at or prior to
the onset of clinical illness and decline in 3–6 months,when it becomes undetectable.
-IgG antibodies to HAV (IgG antiHAV) appear soon after IgM, persist for years after infection, and confer
lifelong immunity.
-IgM anti-HAV has been used as the primary marker of acute infection.
-Elevated titers of IgG anti-HAV in the absence of IgM indicates a past infection.
-Another reliable method to detect acute infection is assaying for viral antigen, which is shed in the feces.
-Antigen is no longer present soon after liver enzymes have reached their peak levels.
-Reverse transcription–polymerase chain reaction (RT-PCR).
-Nucleic acid detection techniques are more sensitive than immunoassays
-The use of vaccine has significantly reduced the incidence of hepatitis A in the United States and has therefore
changed the epidemiology of this infection.
Hepatitis B
-Known as serum hepatitis or long-incubation hepatitis
-The most ubiquitous of the hepatitis viruses
-The highest incidence of acute hepatitis B was among adults aged 25–45 years.
-viable for longer than 7 days on environmental surfaces at room temperature.
-It is detected in virtually all body
-Three major routes of transmission are parenteral, perinatal, and sexual.

Serologic Markers of HBV Infection

-42-nm DNA virus classified in the Hepadnaviridae family. The liver is the primary site of HBV replication.
-Core of the antigen is synthesized in the nuclei of hepatocytes and then passed into the cytoplasm of the liver
cell,
where it is surrounded by the protein coat.

HBsAg is a useful serologic marker in patients before the onset of clinical symptoms because it is present during
the prodrome of acute hepatitis B. The HBsAg is not infectious; its presence in the serum may indicate the
presence of the hepatitis virus.
-HBsAg is the only serologic marker detected during the first 3–5 weeks after infection in newly infected
patients.
-Average time from exposure to detection: 30 days (range, 6–60 days).
-Nucleic acid tests can detect HBV DNA in the serum 10–20 days before detection of HBsAg.

HBcAg is present only in the nuclei of hepatocytes during an acute infection with HBV.
-Chronic infection can test positive for IgM anti-HBc.
-DN-dependent DNA polymerase - required for viral replication and is detectable in serum during the phase of
active viral replication.

Hepatitis B e Antigen, an antigen closely associated with the core of the viral particle, is detected in the serum of
persons
with acute or chronic HBV infection.
-The presence of the e antigen = number of infectious virus particles
= degree of infectivity
-The presence of HBeAg in HBsAgcarriers predicts a severe course and chronic liver disease.
-Presence of anti-HBe antibody in carriers indicates a low infectivity of the serum
-The e antigen is detected in serum only when surface antigen is present

Nucleic acid hybridization or PCR technique is used to detect HBV DNA in the blood; more sensitive
measurement of infectivity and disease progression than serology
HBsAg is the antigen used for hepatitis B vaccination. For nonresponders to vaccination -- HBIG administered
alone

90% recover within 6 months. Others lead to chronic hepatitis.

Four phases based on the virus–host interaction:
 immune tolerance
 immune clearance (HBeAg-positive chronic hepatitis)
 low or non-replication (inactive HBsAg carrier), and
 reactivation (HBeAg-negative chronic hepatitis)

Hepatitis C
-(“non-A non-B hepatitis”)
-Flaviviridae family; Transmitted primarily by blood transfusion of inappropriately screened blood products.
-3% of the world population is infected and most infections become chronic and may
lead to cirrhosis, end-stage liver disease, HCC, and death.
- leading cause of liver transplantation in the US.
- antibody is usually not detected in the first few months of infection but will almost always be present in the later
stages.
-Two laboratory tests are commonly used:
 anti-HCV detection by enzyme immunoassay (EIA) -- initial
 quantitative nucleic acid PCR assays for serum HCV RNA

A positive HCV antibody test result generally indicates that the patient has been exposed to the HCV virus,
this test cannot determine whether the patient is currently infected with HCV or has recovered from HCV
infection.

Anti-HCV positive but HCV RNA negative are recommended to retake the test for HCV RNA on a second
occasion, 3–6 months after the first HCV RNA test.

About 80% of infected patients develop chronic hepatitis. Patients with chronic HCV infection appear to be at
high risk for liver cirrhosis (especially with alcohol consumption).
Most chronically infected patients are asymptomatic and manifest only mild elevations of liver function tests,
especially transaminases.
Treatment: Patients with chronic HCV infection are usually treated with pegylated interferon and ribavirin.

Hepatitis D
-Unique subvirus satellite virus infection; a small, defective RNA-containing virus that cannot replicate
independently but rather requires the HBsAg of HBV for replication.
-Incapable of causing any illness in patients who do not have HBV infection.; HDV virions possess an outer
envelope composed of HBsAg proteins; HBsAg-mediated binding to a cellular receptor helps HDV penetrate the
hepatocyte.
HDV superinfection is likely to become chronic simply because HBV infection is already chronic.
Treatment: Interferon-alpha (longer duration)

Hepatitis E
-Nonenveloped RNA virus that is only 27–34 nm in diameter
-Sole member of the genus Hepevirus in the family Hepeviridae.
-Incubation period: 21-42 days prior to the onset of symptoms.
-The virus in feces and bile by about 7 days after infection
-Nonhuman primates such as pigs, cows, and sheep are susceptible to infection with HEV
-More severe than HAV
-In general, hepatitis E infection is mild, except in pregnant women, in whom it may be a devastating illness.

Acute hepatitis E is diagnosed when the presence of IgM anti-HEV is detected.

EIA and immunochromatography are most convenient for the detection of IgM and/or IgG antiHEV.
Confirmatory: RT-PCR

-No commercially available vaccines exist

-Experimental immune prophylaxis against HEV based on recombinant antigens confer short-term protection
and may be useful for pregnant women in endemic areas and travelers coming into these regions.

Other Forms of Hepatitis

The role of G viruses is currently unclear.
Hepatitis F is an enteric agent that may be transmitted to primates.
Other forms of viral hepatitis, such as TT virus and SEN virus, may exist.
The GB group of flavo-like viruses, GBV-A, GBVB, and GBV-C, are also associated with acute and chronic
hepatitis.
Cytomegalovirus, Epstein-Barr virus, and probably several other agents can also cause hepatitis.