TESTS FOR LIVER

FUNCTION
Update 25 Maret 2020 from FA. Davis
• The clinical laboratory offers several tests for the assessment of liver
function.
• The enzymes alkaline phosphatase, ALT, AST, GGT, and 5′-nucleotidase are
helpful in the assessment of the proper functioning and inflammatory
status of the liver.
• Because the liver is the site for metabolism of carbohydrate, protein, and
lipids synthesis of many proteins
• Conjugation of bilirubin, and detoxification of drugs and other substances
Liver may be assessed by measurement of total and direct bilirubin, total
protein and albumin, cholesterol and triglycerides, and urea and ammonia
• Correlation of laboratory results across time is an indication of
accuracy and appropriateness of results.
• In this case, increased levels of enzymes and bilirubin and lowered
protein correlate with liver disease.
• The extent of the increased alkaline phosphatase and the presence of
increases in both total and direct bilirubin help to specify this liver
disorder as obstructive jaundice
BILIRUBIN METABOLISM
• Bilirubin is a degradation product of the heme portion of hemoglobin.
• Heme is degraded in cells of the reticuloendothelial system, mainly the spleen.
• The protoporphyrin ring of the heme is opened to the biliverdin form and iron
is released.
• Biliverdin is reduced to produce the yellow-pigmented molecule bilirubin.
• The bilirubin molecule, a tetrapyrole, has low solubility in water or plasma.
• When it is released into blood, it is bound to albumin for transport.
• Covalently bound bilirubin is called delta bilirubin.
• When the bilirubin-albumin form reaches the liver, it loses albumin and enters
the hepatocyte.
• Within the hepatocyte, the liver enzyme uridyl diphosphate
glucuronyltransferase (UDPG-transferase) transfers molecules of
glucuronate, a sugar, to the bilirubin molecule
• About 85% of bilirubin is conjugated with two molecules of
glucuronate to form diglucuronate-bilirubin.
• Most of the rest of bilirubin is conjugated with one sugar molecule to
form monoglucuronate-bilirubin.
• The addition of the sugar group increases the solubility of the
molecule.
• Conjugated bilirubin passes into the intestine through the bile duct,
where intestinal bacteria reduce bilirubin to urobilinogen.
• Some urobilinogen may be reabsorbed through the intestinal mucosa
and returned to the portal circulation and the liver.
• The remaining urobilinogen is excreted into urine or oxidized to form
urobilin and excreted in the feces.
• Urobilin is one component in feces giving its characteristic brown
color.
Case 1 ;Hyperbilirubinemia: A
Yellow Serum Sample in a Rack
of Tubes
• The laboratory technologist noticed as he was placing the serum sample cup into
the automated chemistry instrument that the serum appeared to have icterus.
• Although the increased yellow color of the serum could be caused by dye that
has been ingested by the patient, the most common cause of increased
yellowness of the serum is increased bilirubin.
• When the results of the chemistry test for this patient appeared on the screen,
the technologist saw that the total bilirubin for this patient was increased and
that other liver function tests were also abnormal.
• The patient was diagnosed with obstructive jaundice.
• The laboratory results for the patient are typical of the progress of a patient with
this disorder.
Tabel 1.Test Results of the
Patient With the Icteric
Specimen
Test Result Reference range
Total protein (g/dl) 7,4 6.4–8.3
Albumin (g/dl) 4,4 3.5–5.2
Total Bilirubin (mg/dl) 3,5 0.0–1.0
Direct Bilirubin (mg/dl) 2,3 0.0–0.2
ALT (U/L) 220 <45
AST (U/L) 190 <38
ALP (U/L) 374 53–128
GGT (U/L) 178 <55
Amylase (U/L) 94 28–100
• In a patient with obstructive jaundice, the increase in bilirubin is
primarily an increase in conjugated bilirubin.
• The enzymes gamma-glutamyltransferase (GGT), aspartate
transaminase (AST), and alanine transaminase (ALT) may be increased
by as much as five times the normal value.
• Alkaline phosphatase may be increased up to three times the upper
value of the normal range.
• For this patient, both total and conjugated bilirubin levels are
elevated.
• Serum enzymes, including GGT, AST, ALT, and alkaline phosphatase,
are also increased.
HYPERBILIRUBINEMIA
• Bilirubin levels in the blood are increased as the result of several disorders or
conditions.
• These disorders or conditions are categorized into three phases of bilirubin
metabolism, prehepatic, hepatic, and posthepatic.
• Prehepatic hyperbilirubinemia is caused by increased hemolysis and
increased degradation of heme.
• Prehepatic hyperbilirubinemia occurs in patients with sickle cell anemia and
other hemolytic diseases that cause increased destruction of red blood cells
and release of hemoglobin.
• The typical serum bilirubin pattern of prehepatic hyperbilirubinemia is
increased unconjugated bilirubin and normal conjugated bilirubin.
• Hepatic hyperbilirubinemia is generally due to defective transport to
the liver or conjugation of bilirubin in the hepatocytes.
• Disorders of transport into the hepatocytes or conjugation disorders
result in increased unconjugated bilirubin.
• Examples of these conditions include Gilbert’s and Crigler-Najjar
syndromes.
• Gilbert’s syndrome is a hereditary disorder in which there is decreased
bilirubin transport into the hepatocytes.
• Several hepatic hyperbilirubinemic disorders are caused by failure of
the liver to conjugate bilirubin.
• Crigler-Najjar syndrome results from a hereditary deficiency of the UDPG-transferase
enzyme
• Neonatal jaundice is caused by the inability of the immature liver of the newborn to
produce UDPG-transferase.
• A slight increase in bilirubin in the second and third days of life is a normal response.
• Damage to hepatocytes by hepatitis, cirrhosis, toxic substances, and other disorders
can inhibit conjugation as well.
• The typical serum bilirubin pattern of hepatic hyperbilirubinemia is increased
unconjugated and conjugated bilirubin.
• Serum enzymes that indicate hepatocellular inflammation and cellular damage within
the liver, including ALT and AST, are also often elevated.
• Posthepatic hyperbilirubinemia is generally due to a defect in transporting
conjugated bilirubin and bile out of the liver.
• It can involve obstruction of the small canaliculi within the liver, the hepatic bile
duct, and the common bile duct leading to the duodenum of the small intestine.
• Posthepatic hyperbilirubinemia is often called obstructive jaundice.
• Obstruction of the bile flow can be due to gallstones or to scarring and nodules,
such as from cirrhosis or tumors.
• The typical serum bilirubin pattern of posthepatic hyperbilirubinemia is increased
conjugated bilirubin but normal unconjugated bilirubin.
• Serum enzymes that indicate biliary cell damage, including alkaline phosphatase
and GGT, are also often elevated.
Obstructive Jaundice
• Obstruction of bile from the liver may be caused by gallstones in the bile duct or a tumor that
impedes the flow of bile into the intestine.
• Increased total bilirubin in the blood of a patient with obstructive jaundice is usually a
reflection of increased conjugated bilirubin.
• Other effects of obstructive jaundice may be defective excretion of lipid substances through
bile.
• Obstructive jaundice is associated with increased levels of alkaline phosphatase and GGT and
increased serum total and direct bilirubin.
• When the direct bilirubin level is nearing the total bilirubin level, this generally indicates a
posthepatic hyperbilirubinemia such as in obstructive jaundice.
• Sometimes other liver enzymes are elevated as well, indicating hepatic inflammation.
• Ultrasound and other imaging techniques are needed to locate the source of the obstruction.
Neonatal Hyperbilirubinemia:
Why Is the Baby Yellow?
• When a clinical laboratory scientist’s first nephew was born, her family
members told her that the delivery of the full-term infant was normal.
• All clinical signs and laboratory data were normal in the first few days.
• By day three, the infant’s total bilirubin was 5.8 mg/dL and he appeared
jaundiced.
• Home phototherapy was started, along with recording fluid intake, and
the baby wasmonitored daily with serum total bilirubin results.
• Family members wanted an explanation of the cause for this condition and
if this condition likely indicated some inborn liver disorder.
Physiological Jaundice of the
Newborn
• It was noted that neonatal jaundice is typically due to the short-term or
transient immaturity of the liver.
• This causes a short-term delay in ability to produce UDPG-transferase
for conjugation.
• In addition, there is higher turnover of neonatal erythrocytes shortly
after birth in order to replace fetal hemoglobin (Hb F) with hemoglobin
A.
• This causes an increase in supply of heme for degradation to bilirubin.
• This peaks at around 2 to 4 days but may remain elevated for up to 2
weeks
OTHER LABORATORY TEST
RESULTS THAT
CORRESPOND WITH LIVER
DISORDERS
• Since the majority of proteins found in plasma are synthesized by the
liver from amino acids, including serine protease coagulation factors,
prothrombin time, serum albumin, and serum protein electrophoresis
results can be used to indicate declining liver function.
• For example, serum albumin decreases and prothrombin time
becomes prolonged with liver failure.
• Protein levels also reflect other disorders, such as those in which
essential amino acids are not provided by the diet or in which
proteins are lost by the kidneys or gastrointestinal tract.
• Serum contains a large variety of proteins and a large amount of total
protein, averaging 7.0 g/dL in the adult.
• In contrast, protein levels in serum and urine are normally in the
microgram or milligram per deciliter range.
• Methods for measuring proteins in body fluids are based upon the
unique structural properties as well as their relative concentration in
the body fluids.
• There are two main types of proteins, albumin and globulins.
• They are grouped into five classes as determined by their electrophoretic
separation: albumin, alpha1 globulins, alpha2 globulins, beta globulins, and
gamma globulins.
• Serum albumin, at around 4 g/dL, makes up roughly half of the total serum
proteins.
• A simple way to assess the balance between the patient’s albumin and globulins
in serum is to calculate the albumin:globulin, or A/G, ratio.
• Globulins (G) are calculated as albumin (A) subtracted from total serum proteins.
• Albumin is then divided by globulin.
TOTAL SERUM PROTEIN
• Although there are many classic and reference methods for
quantification of serum protein, the biuret reaction has become the
most commonly used method in the clinical laboratory.
• The peptide bonds of proteins react with biuret reagent containing Cu2
ions in an alkaline solution to form a violet color measured at 540 nm.
Sodium potassium tartrate is a component of the reagent and functions
to maintain copper in the correct valence state and in an alkaline
solution, while potassium iodide is present as an antioxidant
The Reaction
Proteins + biuret reagent → violet-colored product (540-nm absorbance)
Interferences
• Marked hyperbilirubinemia and lipemia cause interference unless
corrected with a serum blank.
• Marked lipemia should be removed with acetone-pretreated samples.
• Ambulatory patients exhibit a slight hemoconcentration causing a
physiological increase in serum protein by 0.3 g/dL.
The Specimen
• Serum, free from lipemia and collected without prolonged tourniquet
use, may be used.
• Plasma can be used, but the reference range must be adjusted
upward to account for fibrinogen.
• Plasma should not be used for protein electrophoresis.
Reference Range
• Adult (ambulatory) : 6.4–8.3 g/dL
Interpretation of Total Serum
Protein Levels
• Total serum protein levels are affected by not only changes in one or more of
the individual protein levels, but also by changes in plasma water.
• A variety of conditions cause hyperproteinemia, or increased serum protein.
• Hemoconcentration,or decreased plasma water volume, will cause total
serum protein levels to be increased.
• Dehydration is the usual cause of hemoconcentration, which is secondary to a
variety of conditions including diarrhea, severe vomiting, and water
deprivation.
• Increased total serum protein levels can also occur when there is an increase
in a variety of immunoglobulins following inflammation or infection or a
monoclonal increase in immunoglobulins, such as in multiple myeloma.
• Increased total protein can also result from measuring an unexpected
protein such as fibrinogen.
• Serum is derived from clotted whole blood in which fibrinogen is
removed in the clotting process.
• However, if incomplete clotting occurs before centrifugation, some
fibrinogen can remain behind in the serum specimen.
• Hypoproteinemia, or decreased protein levels in the blood, is often
due to hypoalbuminemia, since albumin is the most abundant single
protein.
• Typical causes of hypoproteinemia are starvation or nutritional
deficiency of essential amino acids, renal loss such as in nephritic
syndrome, gastrointestinal loss such as in enteropathy, or hepatic
failure in which the liver is unable to synthesize proteins.
• Serum protein electrophoresis results can also indicate inflammatory
states of the liver in which there are elevated gamma globulin protein
fractions, especially immunoglobulin A (IgA) and IgM levels.
• In cirrhosis, protein electrophoresis results show that fast-moving
gamma globulins often migrate in the beta to gamma region, causing
a beta-gamma bridge.
Albumin
• Serum or plasma albumin methods are based upon structural characteristics.
• Albumin makes an excellent antigen and can be analyzed by immunoassays such as nephelometry.
This is the method of choice for albumin in cerebrospinal fluid.
• However, other methods such as turbidimetry are also available with chemistry analyzers.
• A simpler method is used when considering typical serum albumin levels.
• The routine method is based upon albumin’s ionic charge at an acid pH and binding to anionic dyes
such bromcresol green (BCG) or bromcresol purple (BCP).
• In these methods, a colored product forms that absorbs at wavelengths significantly different from the
reagent and from typical interfering molecules such as hemoglobin or bilirubin.
• The Reaction
Albumin + BCG pH 4.2 colored product (628-nm absorbance)
• Reference Range
Adult : 3.5–5.2 g/dL
Interpretation of Serum Albumin
Levels
• Albumin levels are affected not only by changes in albumin level, but also by changes in
plasma water volume, in a manner similar to effects on total serum protein.
• In addition to dehydration, fluid redistribution such as in ascites may cause
hypoalbuminemia as well
• Hypoalbuminemia from deficient protein level is due to loss, such as from the
gastrointestinal system in malabsorption or protein-losing enteropathy, from the renal
system as in glomerulonephritis or nephritic syndrome, or from skin due to severe burns.
Hypoalbuminemia can also result from increased catabolism as a result of tissue damage
and inflammation, as found in neoplasms or autoimmunity.
• Decreased serum albumin is also associated with malnutrition and inadequate amino acid
intake.
• Finally, hypoalbuminemia is correlated with declining synthesis in the liver, as associated
with cirrhosis or other situations of liver failure.