LIVER FUNCTION TEST 5.

Storage Function
 Storage site for all fat-soluble
Liver
vitamins and water-soluble
- the chief metabolic organ in the body vitamins
- receive 15 mL of blood per minute  Liver also is the storage depot for
- composed of 2 types of cells: hepatocytes glycogen, which are released when
and Kupfer cells (phagocytes) glucose is depleted
- to abolish liver tissue function, more than
80% of the liver must be destroyed
- synthesizes proteins, coagulation factors, Tests Measuring the Hepatic Synthetic Ability
ammonia, carbohydrates, fat, ketones,
- useful for quantifying the severity of
vitamin A, enzymes, and so on.
hepatic dysfunction

Functions of the Liver

1. Total Protein Determination
1. SYNTHETIC FUNCTION - important for assessing nutritional status
 Secretes plasma CHON, CHO, lipids, and presence of severe disease involving
lipoproteins, clotting factors, ketone the liver, kidney, and bone marrow
bodies and enzymes - total protein and albumin are about 10%
 Produces about 12 grams of albumin higher in ambulatory individuals
daily - fasting not required
 Involves in metabolism of cholesterol into - usually used serum samples
bile acids - avoid hemolyzed samples
- reference values: 6.5-8.3 g/dL
2. CONJUGATED FUNCTION
 Bilirubin metabolism
 200-300 mg of bilirubin is produced daily Methods for Total Protein Determination
in a healthy adult
1. Kjeldahl method
- standard reference method: not commonly
3. DETOXIFICATION AND DRUG
performed method
METABOLISM
- based on measurement of nitrogen
 Serves to protect the body from potentially
content
injurious substances absorbed from the
- it uses serum treated with tungstic acid,
intestinal tract and toxic by-products of
forming protein-free filtrate (PFF)
metabolism
- 1 gram of nitrogen = 6.54 g of proteins
 Ammonia (toxic by-products) is converted
- 15.1% -16.8% = nitrogen content of
to urea in the liver
proteins
- reagent: H2SO4 (digesting agent)
4. EXCRETORY AND SECRETORY
FUNCTION
2. Biuret Method
 Excretion of bile - bile acids or salts,
- most widely used method recommended
pigments, cholesterol
by IFCC
 Cholic acid and chenodeoxycholic acid-
- extensively used in clinical laboratories
bile acids
- can measure down 10 or 15 mg/dL
 - principle: Cupric ion complex the groups  Alpha 2-macroglobulin
involved in the peptide bond forming a  Haptoglobin
violet-colored chelate which is  Beta-lipoprotein
proportional to the number of peptide  Complement C3
bonds present and reflects the total CHON  Fibrinogen and immunoglobulins
at 545 nm
- reagents: Normal SPE Pattern
o Alkaline copper sulfate 1. Albumin (1st fastest band)
o Rochell salt (Na K tartrate) 2. Alpha 1 - Globulin (2nd fastest band)
o NaOH and potassium Iodide - increases as a non-specific response to
inflammation
3. Folin-Ciocalteu (Lowry) Method - glycoproteins, AAT, AAG, THYROXINE
- it has the highest analytical sensitivity BONDING-GLOBULIN (TBG)
- using phosphotungstic-molybdic acid 3. Alpha 2 - globulin (3rd fasted band)
or phenol reagent, and biuret reagent - haptoglobin, AMG, Ceruloplasmin
- oxidation of phenolic compounds such 4. Beta-globulin (4th fastest band)
as tyrosine, tryptophan, and histidine to - transferrin, beta-lipoprotein, hemopexin,
give a deep blue color complement system (C3 and C4)
5. Gamma Globulin (Immunoglobulins)
4. Ultraviolet Absorption Method - slowest band; 5th band
- protein absorb light at 280 nm and at 210
nm
- absorption at 280 nm is due to Abnormal Serum Electrophoretic Patterns
tryptophan, tyrosine and phenylalanine
- the absorbance of proteins at 210 nm is 1. Gamma spike - MM
due to the absorbance of the peptide bonds 2. Beta-gamma bridging - hepatic cirrhosis
at that wavelength 3. Alpha 2-globulin band spike - nephrotic
syndrome
5. Electrophoresis 4. Alpha 1-globulin flat curve - juvenile
- principle: Migration of charged particles in cirrhosis (AAT deficiency)
an electric field 5. Spikes of alpha 1, alpha 2 and Beta
- the single most significant clinical globulin bands - inflammation
application of serum protein
electrophoresis (SPE) is for the
identification of monoclonal spikes of 6. Refractometry
immunoglobulins and differentiating them - Alternative test to chemical analysis of
from polyclonal hypergammaglobulinemia serum total proteins
- myocardial infarction produces a pattern of - Based on measurement of refractive
acute inflammation associated with tissue index due to solute in serum
injury
7. Turbidimetric and Nephelometric Methods
- utilizes sulfosalicylic acid and/or
Major proteins that contribute to electrophoresis: trichloroacetic acid
8. Salt Fractionation
 Albumin - globulins can be separated from albumin
 Alpha 1 –antitrypsin by salting-out procedures using sodium
 salts  Acute viral hepatitis - prolonged
- Sodium sulfate: salt used for analysis PROTIME (cellular damage)
(salting-out)

ALBUMIN
Albumin is soluble in water, moderately
 concentration of albumin is inversely
concentrated salt solution, and concentrated salt
proportional to the severity of the liver
solution.
disease
Albumin is insoluble in saturated salt solution,  decreased serum albumin concentration
highly concentrated salt solution, and may be due to decreased synthesis
hydrocarbon solvents.  Low TOTAL CHON+ LOW albumin =
hepatic cirrhosis and nephrotic syndrome

Globulin is soluble in weak salt solution and

hydrocarbon solvents. Dyes Used for Measurement
Globulin is insoluble in water, saturated salt 1. Bromcresol Green (BCG)- most used dyes
solution, and concentrated salt solution. 2. Methyl orange (MO)
3. Hydroxyazobenzene benzoic acid (HABA)
4. Bromcresol Purple (BCP) - most specific
Other Measurements for Protein Dyes
1. Coomassie Brilliant blue dye is for
detection down of 1 ug of CHON
Albumin/Globulin Ratio
2. Ninhydrin (violet color) - widely used for
detecting peptides and amino acid after  Globulin is increased disproportionately to
paper chromatography albumin in chronic inflammation, MM and
Waldenstroms macroglobulinemia
 If globulin is greater than albumin it is
Increased Total Protein: Malignancy, Multiple known as inverted A/G ratio
Myeloma, Waldenstrom’s Macroglobulinemia  reference value: 1.3-3:1
Decreased Total Protein: Hepatic Cirrhosis,
Glomerulonephritis, Nephrotic Syndrome,
Analbuminemia - hereditary absence of albumin;
Starvation
inability to synthesize albumin
Bisalbuminemia - albumin that has unusual
Prothrombin Time (Vitamin K Response Test) molecular characteristics; presence of two
albumin bands instead of single band in
 Vitamin K is administered
electrophoresis; associated with the presence of
intramuscularly, 10mg daily for 1-3 days
therapeutic drug in serum
 Differentiates intrahepatic disorder
(prolonged protime) from extrahepatic
obstructive liver disease (normal protime)
Tests Measuring Conjugated and Excretion
 Prolonged protime - loss of hepatic
Function
capacity to synthesize proteins
Bilirubin
 - end product of hemoglobin metabolism conjugation of hormones and bilirubin to water-
and the principal pigment in bile soluble forms, and conversion of drugs to
- is also formed from destruction of heme- metabolites for excretion in urine or stool.
containing proteins such as myoglobin
catalase and cytochrome oxidase
- principal pigment in bile that is derived  Jaundice (icterus) - yellow discoloration
from hemoglobin breakdown that occurs when the bilirubin
- produced in the reticuloendothelial system concentration in the blood rises (>2—3
from the breakdown of hemoglobin from nig/dL) and the bilirubin is deposited in
senescent red blood cells (RBCs) the skin and sclera of the eyes.
- bilirubin forms a complex with albumin  Kernicterus - Elevated bilirubin deposits
for transport to the liver in brain tissue of infants, affecting the
- in this form, bilirubin is unconjugated and central nervous system and resulting in
not water-soluble mental retardation.
- bilirubin is conjugated in the hepatocyte
endoplasmic reticulum with glucuronic
acid to form bilirubin diglucuronide Unconjugated Bilirubin vs. Conjugated
(conjugated bilirubin) which is catalyzed
by uridine diphosphate
glycuronyltransferase (UDPGT)
- conjugated bilirubin is water-soluble
- conjugated bilirubin is excreted into the
bile for storage in the gallbladder, secreted
into the duodenum in response to
gallbladder stimulation, and reduced by
anaerobic bacteria in the intestine to
urobilinogen
- some intestinal urobilinogen is reabsorbed;
a portion returns to the liver and some
enters the circulation for excretion in the
urine, whereas the remaining portion in the
intestines is oxidized by anaerobic bacteria Bilirubin
for excretion in the stool as urobilin
- urobilin is an orange-brown pigment that
gives stool its characteristic color
 Liver secretes bile to assist in digestion.
 Bile salts are composed of cholic acid and Reference values:
chenodeoxycholic acid conjugated with  0-0.2 mg/Dl (0-3 umol/L) - conjugated
glycine or taurine. bilirubin
 Bile is stored in the gallbladder.  0.2-0.8 mg/dL (3-14 umol/L) -
unconjugated bilirubin
 0.2-1.0 mg/dL (3-17 umol/L) - total
Detoxification and Drug Metabolism bilirubin
The liver is the primary site in the body for
synthesis of waste products (e.g., urea),
Delta bilirubin
 - conjugated bilirubin tightly bound to such as acute cholangitis or acute
albumin pancreatitis.
- longer half-life than other forms of - characterized by a significantly increased
bilirubin level of conjugated bilirubin in serum,
- helps monitoring the decline of serum increased level of unconjugated bilirubin
bilirubin following surgical removal of in serum, increased conjugated bilirubin in
gallstones the urine, decreased urine and fecal
- computed by using formula: TB-DB+IB = urobilinogen, and stool that appears pale in
Delta bilirubin color.
- reference value: <0.2 mg/dL (<3 umol/L)

3. Hepatocellular-Combined Jaundice
Icterus or hyperbilirubinemia - mechanism: hepatocytes injury caused by
virus alcohol and parasites
- characterized by a yellow discoloration of
- bilirubin assay: Elevated direct and
skin, sclerae and mucus membrane
indirect bilirubin
- clinically evident when bilirubin level
exceed 2 or 3 mg/dL
Derangements of Bilirubin Metabolism
Classification of Jaundice 1. Gilbert’s Syndrome
1. Pre-Hepatic Jaundice - Bilirubin transport deficit
- mechanism: Too much destruction of RBC - Impaired cellular uptake of bilirubin
- bilirubin assay: Elevated indirect bilirubin - Diagnosed in young adults
- seen in hemolytic anemias, spherocytosis, - Affected individuals have no symptoms
toxic conditions, hemolytic disease of the but have mild icterus
newborn caused by Rh or ABO - Elevated B1= <3 mg/dL
incompatibility, and so on - Defect in the ability of hepatocytes to take
- in these cases, the rate of hemolysis up bilirubin; due to transport problem of
exceeds the liver's ability to take up the bilirubin from the sinusoidal membrane to
bilirubin for conjugation the microsomal region; characterized by
- characterized by an increased level of mild increase in serum level of
unconjugated bilirubin in the serum unconjugated bilirubin (1.5-3.0 mg/dL)
2. Crigler-Najjar Syndrome
2. Post-Hepatic Jaundice - Conjugated bilirubin deficit
- mechanism: Failure of bile to flow to the - Infants treated with phototherapy
intestine/ impaired bilirubin excretion
- bilirubin assay: Elevated direct bilirubin
- this is referred to as extrahepatic  Type 1 Crigler-Najjar Syndrome
cholestasis and may be caused by
gallstones obstructing the common bile ✓ deficient of UDPGT
duct, neoplasms such as carcinoma of the
✓ Total absence of B2 production
ampulla of Vater or carcinoma of the
pancreas, and inflammatory conditions ✓ Great danger is kernicterus; bile is colorless
 - causes increased serum level of
unconjugated bilirubin
 Type II Crigler-Najjar Syndrome

✓ Partial deficiency of UDPGT

6. Intrahepatic cholestasis
✓ Small amount of B2 is produced
- may be caused by hepatocyte injury such
as cirrhosis, bile duct injury such as Rotor
Partial or complete deficiency of UDPGT; little, if syndrome, or neoplasms
any, conjugated bilirubin formed, which causes
increased serum level of unconjugated bilirubin
(moderate to extremely elevated). Other Disorders of the Liver
1. Cirrhosis
- result of chronic scarring of liver tissue
3. Dubin-Johnson Syndrome and Rotor Syndrome turning it into nodules
- Bilirubin excretion deficit - may be caused by excessive alcohol
- Hereditary disorders ingestion over a long period of time,
- Blockage of the excretion of bilirubin into hemochromatosis, complication of
the canaliculi hepatitis
- Dark pigmentation of the liver
(accumulation of lipofuscin pigment)
2. Tumors
- Elevated total Bilirubin and B2
 Hepatocellular carcinoma or
- Defective liver cell excretion of bilirubin
hepatoma: primary cancer of the
due to impaired transport in the hepatocyte
liver
of conjugated bilirubin from microsomal
region to the bile canaliculi; characterized  Metastatic liver tumors: Arise from
by increased serum level of conjugated other cancerous tissue where the
bilirubin with mild increase in primary site was of lung, pancreas,
unconjugated bilirubin gastrointestinal tract, or ovary
origin

4. Lucey-Driscoll Syndrome
3. Reye’s Syndrome
- familial form of unconjugated - Cause is unknown, but the symptoms
hyperbilirubinemia include encephalopathy, neurologic
- circulating inhibitor of bilirubin abnormalities including seizures or coma,
conjugation and abnormal liver function tests due to
hepatic destruction
- Occurs mainly in children, usually after a
5. Neonatal Physiological Jaundice viral infection (varicella or influenza) and
aspirin therapy
- level of UDP-glycuronyltransferase is low
at birth; takes several days for the liver to
synthesize an adequate amount of the
4. Drug-Related Disorders
enzyme to catalyze bilirubin conjugation
 - Drugs, including phenothiazines, ▪ Not falsely elevated by hemolysis
antibiotics, antineoplastic drugs, and anti-
▪ Coupling accelerator: Caffeine Sodium
inflammatory drugs such as
Benzoate
acetaminophen, may cause liver damage.
▪ Buffer: Sodium Acetate
▪ Ascorbic acid - terminates the initial reaction
IMPORTANT NOTES!
and destroys the excess diazo reagent
 Free from hemolysis and lipemia (serum)
▪ Final reaction: (+) pink to blue azobilirubin
 Sample should be stored in a dark and
measured ASAP or within 2-3 hours after
collection
Direct spectrophotometry: For newborns,
 Serious loss of bilirubin occurs after
bilirubin concentration is read directly by
exposure to fluorescent, indirect, and
spectrophotometry and concentration is
direct sunlight
proportional to absorbance at 455 nm
 >25 mg/L (VISIBLE icterus occur)
 Bilirubin standard solution is usually made Sources of error: Hemolysis, lipemia; avoid
from B1 exposure to sunlight and fluorescent lighting
o Conjugated bilirubin - the fraction
that produced a color in aqueous
solution Reference ranges
o Unconjugated bilirubin - the
▪Infants: Total bilirubin 2-6 mg/dL (0–1-day, full
fraction that produced a color only
term)
after the addition of alcohol
▪Adults: Total bilirubin 0.2-1.0 mg/dL; Indirect
bilirubin 0.2-0.8 mg/dL
Bilirubin Assay
 PRINCIPLE: Van den Berg Reaction -
diazotization of bilirubin

IMPORTANT NOTES!
A. Evelyn and Malloy Method
 Adding a methanol or caffeine solution is
▪ Coupling Accelerator: Methanol
to allow indirect bilirubin to react
▪ Diazo Reagents: (solubilize) with the color reagent.
 Total bilirubin is measured 15 minutes
▪ Diazo A = 0.1 % Sulfanilic Acid +HCl after adding methanol or caffeine solution
▪ Diazo B= 0.5% Sodium Nitrite  Measurement of total bilirubin involves
solubilization of the unconjugated form
▪ Diazo Blank= 1.5% HCl before chemical quantitation
Final reaction: (+) pink to purple azobilirubin  Bilirubin absorbs light maximally at 450
nm and imparts yellow color to amniotic
fluid.
B. Jendrassik and Grof  The binding capacity of albumin for
unconjugated bilirubin is the basic defense
▪ Popular technique for the discreet analyzer
 for prevention of kernicterus in HDN and - absence of this substance in urine or stool
infantile jaundice denotes complete biliary obstruction
 Unbound bilirubin (free bilirubin) may - specimen for testing: 2- hour freshly
cause blood-brain barrier resulting to collected urine and freshly collected stool
kernicterus (avoid photo exposure)
 Measurement of B2 is a sensitive and - method for testing: Erlich’s method (p-
specific marker for hepatic and post dimethyl aminobenzaldehyde reagent)
hepatic jaundice because it is not elevated - reference values: 0.1-1.0 Ehrlich units/ 2
by hemolytic anemia. hours or 0.54 Ehrlich unit/day-urine
 In adults, cholelithiasis (presence of gall o 75-275 Ehrlich units/100g of feces
stones) is the most common cause of or
hyperbilirubinemia. o 75-400 Ehrlich units/24 hour- stool
 Obstruction of the biliary tract also causes
elevated total cholesterol due to block in
the normal excretion of cholesterol in bile. Urine Urobilinogen Assay
Urobilinogen + p-dimethyl aminobenzaldehyde -
red colored complex
 Sources of error: Oxidation will occur if
urine is allowed to stand; other compounds
react, such as porphobilinogen
 Clinical significance :
a. In posthepatic obstruction, urobilinogen
formation is decreased because of
impaired bilirubin excretion into the
intestines. This is evidenced by a clay-
colored (partial biliary obstruction) or
chalky white stool (complete biliary
obstruction).
b. Increased urine urobilinogen is
associated with hemolytic disease and
Bromsulfonthalein Dye Excretion Test
hepatocellular disease, such as hepatitis.
- test for hepatocellular function and c. Reference range urine urobilinogen:
potency of bile duct; rarely used 0.1-1.0 Ehrlich units/2 hr.

Urobilinogen
Tests for Detoxification Function
- colorless end product of bilirubin
- involves enzyme test and ammonia test
metabolism that is oxidized by intestinal
bacteria to the brown pigment urobilin
- collective term for stercobilinogen,
Enzyme Test
mesobilinogen, and urobilinogen.
- it is either excreted in urine and feces, or - any injury to the liver that results in
reabsorbed into the portal blood and cytolysis and necrosis causes the liberation
returned to the liver of various enzymes.
 - used to assess the extent of liver damage - The liver normally removes most of this
and to differentiate hepatocellular NPN via the portal vein circulation and
(functional) from obstructive (mechanical) converts it to urea, then eliminated by the
disease. kidneys (urine)
- enzymes are often the only indication of - Elevated levels are neurotoxic and are
cell injury in early or localized liver often associated with encephalopathy
disease - Clinical Significance: for the diagnosis of
- ALP, aminotransferases (ALT, AST), hepatic failure and Reye’s syndrome
5’NUCLEOTIDASE, GGT, OCT, LAP, - increases pH of Central Nervous System:
LDH are the enzymes secreted by the liver in severe liver disorder, it accumulates and
reaches the systemic circulation, which is
then converted to glutamine in the brain,
Markers for hepatocellular necrosis thus compromising the Kreb’s Cycle
leading to coma due to lack of ATP for the
a. ALT: Most specific for hepatocyte injury
brain
b. AST: Less specific than ALT; significant
- increased levels: Reye’s Syndrome,
presence in other tissues
cirrhosis, hepatic come, acetaminophen
c. LD: Least specific; significant presence in
poisoning
other tissues
- Reference value: 11-35 mmol/L (19-60
ug/Dl)
Markers for cholestasis
a. Alkaline phosphatase IMPORTANT NOTES!
b. Gamma-glutamyl transferase
 Smoking is a source of contamination
(patient and phlebotomist) which leads to
elevated concentrations
Other Tests to Assess Liver Disorders
 Prolonged standing of the specimen rises
a. Total bilirubin, direct bilirubin ammonia level due to enzymatic
(conjugated), indirect bilirubin deamination of labile amides like
(unconjugated) glutamine
b. Albumin  Plasma levels of ammonia are not useful in
c. Ammonia the study of kidney diseases.
d. AFP  Preferred specimen: arterial blood (venous
blood is not recommended, if used,
tourniquet should be used minimally, and
Ammonia fist clenching and relaxing avoided during
collection)
- Arises from deamination of amino acids,
 Specimen requirements: Heparin or EDTA
which occurs mainly through the action of
plasma (fasting); plasma/serum kept in ice
digestive and bacterial enzymes (bacterial
water immediately; hemolysis should be
proteases, ureases and amino oxidase) on
avoided
proteins in the intestinal tract
 Methods for testing: Berthelot reaction,
- It is also released from metabolic reactions
Glutamate dehydrogenase
that occur in skeletal muscles during
exercise