LIVER
FUNCTION TEST
The liver is a large and complex organ
found in the upper quadrant of the body. It is beneath and attached to the
diaphragm and protected by the lower rib cage. The liver accounts for approximately
2.5% of an adults body weight. Weighing between 1200–1600 g, it is unequally
divided into lobes by the falciform ligament, the right lobe being about six
times larger than the left lobe. The lobes have no functional significance, and
there is free communication between all portions of the liver.
The liver is a very vascular organ with
approximately 1500 ml of blood per minute passing through it. The liver is
unusual in that it receives a dual blood supply. Blood rich in nutrients and
other absorbed substances from the gastrointestinal tract is carried to the
liver by the portal vein. Even though the portal vein contributes 80% of the
total blood volume of the liver; it supplies only 40% of the oxygen. The
hepatic artery, branching from the abdominal aorta, is the primary supplier of
oxygenated blood to the liver. To complete the hepatic circulation, blood is
drained form the liver by a collecting system of veins that empties into the
hepatic veins and ultimately into the inferior vena cava.
The liver cells or the hepatocytes
The liver lobule is the basic
microscopic unit of the liver and is responsible for all metabolic and
excretory functions of the liver. Each lobule is roughly hexagonal in shape
with four to six peripherally located portal triads, numerous columns of
hepatic parenchymal cells, a continuous system of blood carrying sinusoids.
1. Kuppfer cells – a special endothelial cell which are
phagocytic and are part of the reticuloendothelial system of the body.
Endothelial cells – are flattened cells that line the
sinusoids and function as filters to prevent the passage of large molecules
into the parenchymal cells.
2. Parenchymal cells – performs the metabolic,
detoxification, excretory and synthetic functions associated with the liver and
are responsible for the regenerative properties of the liver. It accounts for
60–80% of liver volume and has an average life span of 150 days in experimental
animals.
Components of parenchymal cells and its
functions:
a. Golgi apparatus
(1)
Assembles
and transports lipoproteins and
glycoproteins.
(2)
Secretes
albumin and bilirubin.
b. Lysosomes
(1) Contain hydrolytic enzymes
(2) Metabolism of metals
c. Microbodies
(1)
Respiration
(2)
Liquid
and purine metabolism
(3)
Gluconeogenesis
(4)
Detoxification
of alcohol
d. Mitochondria
(1)
Energy
source
(2)
Oxidative
phosphorylation
(3)
Oxidation
of fatty acids
e. Endoplasmic reticulum
(1)
Synthesis
of albumin, coagulation factors,
cholesterols and bile acids
(2)
Metabolism
of drugs and steroids
(3)
Conjugation
of bilirubin
(4)
Deposition
of glycogen
(5)
Glycosylation
of proteins
(6)
Metabolism
of fatty acids, phospholipids and
triglycerides
(7)
Calcium
homeostasis
Functions of the liver:
1. Metabolic function:
a. Carbohydrates
b. Lipids
c. Amino acid and proteins
d. Bilirubin
e. Hormones
f. Cholesterol
2. Excretory function:
a. Bile acids
b. Cholesterol
c. Bilirubin
3. Hematologic function:
a. Production of coagulation factors
b. Production of red blood cells in the
fetus
4. Detoxifying function:
a. Bilirubin
b. Ammonia
c. Alcohol
d. Drugs
5. Storage function:
a. Glycogen
b. Lipids
c.
Amino acids and proteins
d.
Iron
e.
Copper
f.
Vitamins
6. Immunologic function:
a. Phagocytosis to clear bacteria and other
foreign substances
b. Secretion of IgA
c. Humoral defenses
Liver secretions:
1. Bile – is a greenish–yellow liquid secreted by the liver which
contains water, bile salts, bile pigments and cholesterol averaging 600 cc a
day. The bile is transported to the common bile duct and then to the gall
bladder where it is concentrated and emptied into the small intestines. It is
essential for digestion of fats.
2. Bile pigments – manufactures from the products formed
by the breaking down of hemoglobin.
3. Bile salts – most useful constituent of bile not
excreted from the body but are reabsorbed almost completely and recirculate
back to the liver through an enterohepatic pathway. Bile salts normally
function in the emulsification of dietary fats, the activation of the lipases
and in the absorption of lipid through the intestinal mucosa.
4. Bile acids – are catabolic products of cholesterol
formed in the liver. These are either conjugated with glycine or the sulfur
containing compound, taurine to form bile salts.
METABOLIC FUNCTION OF THE LIVER
It is presumed that patients with
hepatic diseases may have hypoglycemia, decreased carbohydrate tolerance and
decreased hepatic glycogen stores.
I.
Carbohydrate metabolism
A. Galactose
Tolerance Test
This is based on the ability of the
normal liver to convert galactose to glycogen. The galactose that is carried to
the liver cells from the intestinal tract is normally converted into glucose,
which is then further converted to glycogen. If galactose is subjected into the
blood, the speed of removal of this sugar is related to the integrity of the
liver and the normal functioning of its cells. In liver diseases, little or not
conversion of galactose to glycogen takes place.
Preparation
of the patient for:
a.
Oral Galactose Tolerance Test
The patient is given 40 g of galactose
in about 200 ml of water, which may be flavored with lemon juice. Blood samples
are drawn at 30 and 60 minutes after giving the galactose. All urine specimens
voided within the 5–hour period after ingestion are collected and combined.
Normal
values:
40–60 mg/100 ml
in 30–60 minutes
b.
Intravenous Galactose Tolerance Test
One ml of the 50% galactose solution per
kilogram body weight (0.5 g/kg if less concentrated solutions are used) is
injected by a physician under proper clinical condition. A blood sample is
drawn 60 minutes after the completion of the test.
Procedure for galactose determination:
A method for determination of galactose
in blood or urine ordinarily involves removal of glucose by fermentation with
yeast or by treatment with glucose oxidase.
The concentration of the remaining sugar
is determined by the galactose oxidase method and is expressed as galactose.
Normal values:
Should not exceed 42mg/100 ml after 60
minutes
II.
Protein metabolism
Deamination and transamination of amino acids, urea
formation and synthesis of prothrombin and many of the plasma proteins is dependent
on normal liver function. In protein metabolism, an extensive impairment or
destruction of liver cells is required before abnormal function can be clearly
demonstrated.
Test for protein
metabolism:
A. Total protein, A/G ratio (see discussion
on Lecture #10: Proteins)
B. Flocculation and Turbidity Tests
C. Electrophoresis
Flocculation and
Turbidity Tests:
Generally, the response to these test depend on the state
of balance between the stabilizing and precipitating factors in the serum. The
precipitating factors include gamma globulins and lipoproteins such as
betaglobulins. The stabilizing factors are albumin and alpha–1–globulin. In
normal serum, the distribution of the protein components is such that the
stabilizing factors prevent turbidity or flocculation when any flocculation or
turbidity tests are carried out.
a.
Cephalin Cholesterol Flocculation Test
(CCFT) – Hanger’s method
Serum from patients with liver cells
impairment will react with the cephalin–cholesterol suspension to produce a
flocculant precipitate, which is probably an alpha or beta–globulin–cholesterol
complex.
Normal blood serum does not produce
flocculation due to the inhibitory action of the albumin or globulin. A blood
serum will flocculate a colloidal suspension of cephalin–cholesterol due to
increased gamma–globulin and decreased albumin.
Reagents:
1. NSS
2. Ether, anesthetic grade
3. Cephalin–cholesterol stock solution
(prepared from the brain of the sheep)
To the cephalin–cholesterol mixture, add
5 ml of ether. Allow the solution to stand for several hours to obtain complete
solution of the material and keep tightly stored in a refrigerator.
4. Cephalin–cholesterol suspension (working
standard)
Heat 35 ml of freshly prepared distilled
water to 65–70oC, add slowly with constant stirring 1 ml of the
clear stock solution. Heat gently and allow to simmer until the volume is
reduced to 30 ml. A stable milky solution should result and it should be cooled
to room temperature before use. When stored in a refrigerator, the emulsion is
stable for two weeks.
Procedure:
Serum NSS Emulsion
Unknown
0.2 ml 4 ml 1 ml
Reagent
control 4
ml 1ml
Positive
or negative 0.2 ml 4 ml 1 ml
control
Mix gently by inversion and let stand in
the dark at room temperature for 24–48 hours.
To shorten the time required for the
test, some laboratories read the tubes after 4 hour incubation at 37oC.
Examine
the tubes and judge the reaction as follows:
Negative – no flocculation or precipitation
1+
reaction – slight
flocculation or precipitation
2+
reactions – definite
flocculation or precipitation
3+
reaction – almost
complete precipitation with a somewhat cloudy supernatant fluid
4+
reactions – complete
precipitation with a clear supernatant fluid
Normal
values:
Serum from
normal individuals give negative or 1+ reaction
Clinical Significance:
CCFT responds readily to serum specimens
containing increased gamma–globulin and decreased albumin. This condition is
common in a high percentage of cases of viral hepatitis, cirrhosis and hepatic
necrosis and occurs less frequently in post–hepatic obstruction. The test is
very sensitive to qualitative changes in serum albumin, which may explain its
rapid response to acute hepatitis.
The important application of this test
is the differentiation of parenchymal liver disease from early obstructive or
hemolytic jaundice because CCFT is elevated in liver disease, usually unaffected
in the two latter cases.
Notes:
1. Only milky smooth emulsions should be
used in the test.
2. Use carefully cleaned glassware rinsed
with distilled water.
3. Use fresh serum and protect the reaction
from heat and light.
4. Use fresh distilled water to prepare
reagents.
b.
Zinc Sulfate Turbidity Test
Serum from patients with high
gamma–globulin (liver disease) will produce varying degree of turbidity when
mixed with a dilute solution of zinc sulfate in a barbiturate buffer of pH 7.5.
Procedure:
0.2 ml serum
6ml zinc sulfate
reagent
Stopper, mix thoroughly and read the
absorbance against a reagent blank
Use a standard
curve or compute
ZnSO4 turbidity units = A or
O.D. x K
Normal values: 2–12 units
c.
Thymol Turbidity Test
Serum from patients with liver disease
will produce definite turbidity when mixed with a thymol solution in
barbiturate buffer. The turbidity is caused by the precipitation of a globulin
thymol–phospholipid complex. It is also observed in serum with increased
betaglobulin and with lipemic sera.
Procedures:
1. Shank–Hoagland
0.1
ml
serum
1.0
ml
thymol buffer
Mix thoroughly and let stand for 30
minutes at 25–28oC
Mix again and read absorbance against a
reagent blank.
Use a standard curve or compute.
Thymol turbidity units = A or O.D. x K
Normal values: 0.5 Shank–Hoagland units
2. Maclogan
Serum is mixed with thymol buffer in the
proportion of 1:10 and
compared with standard solutions with concentrations in Maclogan units.
For comparison, 1 Maclogan unit = 2
Shank–Hoagland units
Clinical significance:
TTT is affected in increased
gamma–globulin, lipids and beta–globulins and decreased albumin. The test does
not respond as rapidly to viral hepatitis as CCFT but the increase in thymol
turbidity persists at times longer than an abnormal CCFT.
Increased
in: infectious hepatitis, nephrosis, cirrhosis and
diabetes
Decreased
in: obstructive jaundice without liver involvement
Notes:
1. Thymol reagent should be clear and
colorless and must be adequately mixed with serum during the test.
2. pH should be 7.55 as this is more
sensitive than pH 7.8.
3. Reaction should be in a water bath at 25oC
or above. Turbidity decreases as temperatures rises.
4. Test should not be done on lipemic sera
as it will give false positive elevations.
5. Blood should be withdrawn in the fasting
state.
The advantages of TTT over the CCFT are:
1. TTT is the last to return to normal in
liver disease.
2. The turbidity in TTT can be accurately
measured with a photoelectric colorimeter whereas the CCFT is graded only.
Other flocculation tests:
1. Colloidal Gold Test
2. Takata–Ara test (HgCl2 as
precipitating agent)
3. Cadmium Sulfate Test
III.
Bilirubin metabolism
A. The bilirubin of aged or damaged red
blood cells is converted by a complex series of reactions to the bile pigments,
bilirubin.
B. Bilirubin is then transported from the
extrahepatic sources as a bilirubin–albumin complex into the liver (hepatic
sinusoids).
C. In the liver, the protein is separated
from the complex and bilirubin is converted into bilirubin digluoronide by the
reaction with uridine diphosphate gluconate catalyzed by the enzyme system,
UDP–gluconyl transferase.
D. The glucoronide, along with some free
bilirubin, is excreted into the bile, passes into the small intestines and is
exposed to the reducing action of the enzyme of anaerobic bacteria.
E. The reduction products:
mesobilirubinogen, stercobilinogen and urobilinogen collectively known as
urobilin, are first formed in the colon, then a portion of the urobilinogen is
absorbed into the portal circulation and returned to the liver.
F. The normal liver removes all but a small
amount of urobilinogen from the blood, probably oxidizes some of it to
bilirubin and excretes both into the bile for a return trip to the colon.
G. The urobilinogen remaining in the colon
are excreted in the stool after being oxidized to form urobilin (stercobilin),
an orange brown colored pigment.
The reaction is summarized
as follows:
heme
oxygenase
biliverdin
biliverdin
reductase
bilirubin
(B1)
attaches
to albumin
liver
UDP
– glucuronyl transferase
(uridine diphosphate)
bilirubin
diglucoronide (B2)
bile
intestine
(bacterial flora)
reabsorbed
by the stercobilin
enterohepatic
circulation (stool)
(enterohepatic
cycle)
urobilin
(urine)
The Van den Bergh
Reaction
Ehrlich described the coupling reaction of bilirubin with
diazotized sulfanilic acid to form a blue pigment in strongly acid or alkaline
solutions. Van den Bergh applied this color reaction to the quantitative
determination of bilirubin and reported the effect of alcohol on the rate of
coupling reaction. According to the reaction of bilirubin with Ehrlich’s
reagent (diazotized sulfanilic acid).
Van den Bergh classified bilirubin present in the serum
into:
1. Direct–reacting bilirubin or conjugated
bilirubin or bilirubin diglucuronide or B2 – water soluble.
2. Indirect–reacting or unconjugated
bilirubin or B1 – alcohol soluble.
Comparison of properties
of Direct and Indirect bilirubin:
Direct bilirubin Indirect bilirubin
1.
Structure bilirubin
diglucuronide bilirubin
(loosely attached
attached to albumin)
2.
Solubility
in:
a.
water
soluble
insoluble
b.
alcohol
soluble
soluble
3.
Diffusibility
into tissues good poor
4.
Reaction
to Van den Bergh direct
indirect
5.
Presence
in urine present
absent
6.
Toxicity
non–toxic
toxic
Synonyms of Bilirubin 1: Synonyms
of Bilirubin 2:
1.
Unconjugated
bilirubin 1. Conjugated bilirubin
2.
Water
insoluble/Non–polar bilirubin 2. Water soluble/Polar bilirubin
3.
Indirect reacting
bilirubin 3. Direct reacting bilirubin
4.
Hemobilirubin 4. Cholebilirubin/Cholestatic bilirubin
5.
Free bilirubin 5. Prompt bilirubin
6.
Prehepatic bilirubin
6. Post–hepatic bilirubin
7. One–minute
bilirubin
Methods of bilirubin
determination:
General principles:
The chemical
methods for quantitation estimation of
bilirubin in
serum are based on diazotization of the
bilirubin and
measurement of the azo dye.
1.
Malloy and Evelyn (based on Van den
Bergh reaction)
Bilirubin in serum is coupled with diazotized
sulfanilic acid to form a pink or purple compound, azobilirubin, the intensity
of color which is proportional to the bilirubin concentration in the serum.
Direct bilirubin reacts with diazo
reagent in aqueous solution to form a color within one minute, after which the
mixture is read against a reagent blank. The subsequent addition of alcohol
accelerates the reaction of all forms of bilirubin in the serum and a value for
total bilirubin is obtained after allowing the specimen to stand for 15–30
minutes. This represents the total bilirubin which is the sum of the bilirubin
diglucoronide (direct) and the unconjugated bilirubin. Indirect–reacting
bilirubin is determined by difference.
Composition of Ehrlich’s diazo reagent:
a. Diazo A –
0.1% sulfanilic acid
b.
Diazo B –
0.5% sodium nitrite (NaNO2)
c. Diazo blank – 1.5% hydrochloric
acid (HCl)
Modification of Evelyn Malloy method:
a. Thamhauser–Andersen modification
The direct reaction is made on the serum
and the indirect on the alcoholic extract separated from serum after it has
been precipitated by ethyl alcohol and ammonium sulfate.
b. Anino, Watson and Ducci
2.
Jendrassik and Grof
The diazotization is accelerated by
caffeine and sodium benzoate at a strongly alkaline pH for the determination of
total bilirubin. Alkaline copper tartrate is added producing a green color
instead of a blue color which should be the color of diazotized bilirubin in
alkali; but due to the yellow color of the reaction of the diazo reagent and
caffeine mixture, a green color develops. Direct bilirubin is determined 2
minutes after the addition of diazo reagent. The diazotization is terminated by
the addition of ascorbic acid.
Modification of Jendrassik and Grof
method:
a.
Stoner and Wiseberg method
Principle:
Proteins in
serum are eliminated with saturated
ammonium
sulfate (NH4)2SO4 and then
bilirubin is
reacted with diazotized sulfanilic
acid forming
a blue azobilirubin using
alcoholic HCl
as coupling promoter.
Reagents
and results:
1.
Saturated
Ammonium Sulfate – protein
precipitant
2.
Alcoholic HCl – coupling promoter
3.
Ehrlich’s diazo reagent – color reagent
4.
Blue azobilirubin at acid pH – end product
and
color
b.
Alkaline Methanolysis Method
The most accurate method for measuring
bilirubin which involves the formation methyl esters and determination of the
products by High Performance Liquid Chromatography (HPLC). This method is used
in researches only.
Advantages of this method over Evelyn Malloy:
a. Less sensitive to variation in pH,
protein and hemoglobin concentrations in the patient’s sample.
b. Forms minimal turbidity during the
reaction.
c. Sensitive enough to produce sufficient,
reliable color even with very low concentrations of bilirubin.
Precautions in bilirubin determination:
a. Hemolysis decreases the reaction of
bilirubin with diazo reagent, producing falsely low concentrations and lipemia
causes error in the spectrophotometric measurements.
b. Bilirubin is both light and temperature
sensitive. Allowing serum or plasma to be exposed to fluorescent or natural
light reduces bilirubin values by 10% within 30 minutes.
c. Samples are stable for one week in a
dark refrigerator and for three months in a freezer.
d. The patient must fast 8–14 hours to
prevent lipemia.
3.
Thin Film EKTACHEM analyzer compose of:
a. Spreading Layer/Reaction Layer – contains Triton x–100 surfactant,
diazonium salt and dyphylline as accelerator. This layer separates the
unconjugated bilirubin from albumin and contains all of the necessary
components for the quantitation of bilirubin.
b. Control Layer – buffered mordant layer that
stabilizes the azo derivatives produced in the reaction layer and increases the
sensitivity of the assay.
c. Non–reactive transparent support
When the bilirubin in the sample come
into contact with the reagent on the slide, a spectral change occurs. The
reflectance densities of the azo derivatives of all bilirubin fractions are
then measured at 540 nm reflects bilirubin concentrations and measurement at
460 nm is used to correct for spectral interferences. To differentiate
conjugated from unconjugated bilirubin, another type of dry chemistry slide is
used with four layers:
a. Spreading layer – allows uniform dispersal of sample
but also contains caffeine, surfactants and sodium benzoate to disassociate the
unconjugated bilirubin from albumin.
b. First masking layer – where bilirubin removed from albumin
migrate.
c. Second masking layer – uses selective filtration to trap
many large molecules. This layer removes hemoglobin, albumin bound delta
bilirubin, lipids and lipochromes.
d. Registration layer – where the conjugated and unconjugated
bilirubin bind to mordant. Two separate reflection density measurements are
made, one at 400 nm for unconjugated bilirubin and another at 460 nm for
conjugated bilirubin.
Normal values:
Direct–reacting
= 0 – 0.2 mg%
Indirect–reacting
= 0.2 – 0.8 mg%
Total
bilirubin = 0.2 – 1.0 mg%
Interpretation:
Direct
Indirect
Obstructive increased –
Hemolytic – increased
Hepatic increased
–
The Icterus Index
This is a measure of the degree of ictresia or
yellowishness of serum or plasma in cases of jaundice. The degree of
yellowishness is due to hemobilirubin and cholebilirubin.
Principle:
Serum or
plasma is diluted with NSS or sodium
citrate
solution until the color of the specimen
matches with
that of a reference standard. The
standard used
is a 1: 10,000 dilution or 0.01%
potassium
dichromate.
Methods:
1. Muellengracht – 0.85% - 0.90% saline as
diluent
2. Newberger –
sodium citrate as diluent
a. Precautions:
(1)
Specimen
with visible lipemia or hemolysis are unsatisfactory hence, fasting is
necessary.
(2)
Lipochrome
pigments such as carotene are detected with this method, therefore, foods such
as carrots should be avoided the day before the test.
b. Normal values: 6
– 8 Icterus units
IV.
Lipid metabolism
The lipid plays a major role in lipid metabolism. It is
the organ that is involved in the complex transportation of lipid material
between the blood and the bile. It is an important site of synthesis of fatty
acids, ketone bodies and cholesterol esters, phosphatides and lipoproteins.
The typical profile seen is an increased level of
triglycerides and fatty acids, decreased levels of cholesterol esters and the
accompanying alterations in lipoprotein concentrations. Many of these
abnormalities can be attributed to the deficiency of two enzymes of liver
origin: lecithin–cholesterol acyltransferase (LCAT) and hepatic triglyceride
lipase.
The appearance of lipoprotein X as an indicator of
cholestasis provides a specific evaluation of liver dysfunction. Lipoprotein X
contains free cholesterol and phospholipids and has albumin as its primary
apoproteins.
(Methods of lipid quantitation is discussed on Lecture #5)
EXCRETORY FUNCTION OF THE LIVER
Exogenous dye tests have traditionally
been used to test the liver’s detoxification and excretion ability. Dye tests
evaluate first the liver’s ability to transport exogenous substances into the
hepatocytes, then its ability to metabolize the substance, usually by
conjugation to make it more soluble and finally its excretion into bile. These
tests may provide a sensitive picture of the functioning of the liver as a
whole.
A. Bromsulfonphthalein
Dye Excretion Test
It is the most sensitive test for liver
function. It is a valuable test in the detection of parenchymal liver dosage
from any cause before the appearance of jaundice.
Principle:
A measured amount of dye is injected
intravenously. Normally, this dye is removed from the blood by the parenchymal
cells in the liver, excreting it in the bile. Only around 5% of the dye
injected is retained in the blood and this is detected based on the fact that
the dye is colorless in acid and reddish purple in alkaline solution.
The rate of removal of BSP and its
excretion into the bile depends on several factors: the blood level of the dye,
the hepatic blood flow, the condition of the liver cells and the potency of the
bile ducts.
Procedure:
1. To avoid lipemia, the test is usually run
in the morning on the patient in the fasting state.
2. Take the weight of the patient in pounds
and compute for the amount of dye.
a.
Rosenthal method
2 mg/kg body weight dose – 30 minutes = weight in lbs.
= ml of 5% BSP solution
55
b.
McDonald method
5 mg/kg body weight dose – 45 minutes = weight in lbs.
= ml of 5% BSP solution
22
3. Inject into an arm vein the dye from 39
– 60 seconds and start timing as soon as the injection is completed.
4. Extract blood from the opposite arm vein
at the end of the prescribed time.
5. Separate the serum. Icteric, lipemic or
grossly hemolyzed serum should not be used. Treat the samples as follows:
Blank Unknown
Serum 0.5
ml 0.5 ml
Water 2.5
ml 2.5 ml
0.1
N HCl 3.0 ml –
0.1
N NaOH – 3.0
ml
· Sodium p–toluene sulfonate is added to
the alkaline buffer to release the BSP dye bound to albumin, thus diminishing
the effect of proteins on the color of the dye.
6. Read against blank at 575 nm; read the
value on a curve.
Normal values:
Less than 5% of the dye should be obtained after
45 minutes (McDonald method)
No dye in 30 minutes (Rosenthal method)
Sources
of error:
1. Care must be taken so that all of the
dye enters the vein since it is quite irritating to the tissue: Allergic
reactions and anaphylactic shock.
2. In post prandial cases, the removal of
the dye is speed up due to increased
hepatic blood flow and bile excretion.
3. BSP excretion is increased (less
retained) in hypoalbuminemia due to the reduced albumin binding capacity of the
dye.
4. In proteinuria, the dye is lost thru the
urine since it is albumin bound.
B. Indocyanin
Green (ICG) Dye Excretion Test
The ICG is similar to BSP test except
that tricarbocyanine dye is not conjugated in the liver as is BSP. Almost all
of the dye injected is recovered in the bile. Dependence on hepatic blood flow
results in a decreased clearance rate in patients with conditions characterized
by decrease myocardial output. The dosage of dye injected is 0.5 mg per
kilogram of body weight and a normally functioning liver clears 28% (+/– 3%) of
the dye per minute from the circulation.
Dichromatic Ear Densitometry
The Dichromatic Ear Densitometry is
attached to the outer ear, while one photocell detects the concentration of the
dye and a second photocell compensates for changes in hematocrit, oxygen saturation
and blood volume. This provides a noninvasive but accurate evaluation of
arterial levels of the ICG dye.
CONJUGATION AND DETOXIFICATION FUNCTION
OF THE LIVER
The liver by means of conjugation is
able to convert many toxic substances into non–toxic compounds, change active
drugs into inactive conjugates, and alter the solubility of metabolites by
esterification or conjugation to assist in normal excretion. Glycine, glucuronic
acid and sulfates are often used as conjugating agents.
A. Hippuric
Acid Test
Quick (1933) introduced the hippuric
acid test as a test for liver function. Benzoic acid in the form of sodium
benzoate is conjugated with glycine to form hippuric acid for excretion by the
kidney.
Preparation of the patient:
1.
Oral Test
a. Patient must be in the postabsorptive
state
b. After emptying the bladder, give 6 g
sodium benzoate dissolved in about 200 ml of water.
c. Urine is collected for a 4–hour period.
2.
Intravenous test
a. A sterile solution containing 1.77 g of
sodium benzoate in 20 ml water is slowly injected by a physician after the
patient has emptied his bladder and drank a glass of water.
b. The urine is collected for one hour
after the injection with complete emptying of the bladder by the patient.
Advantages of Intravenous
test:
a. Avoidance of nausea or vomiting that may
result from the oral route.
b. Decrease time required for the test.
c. Elimination of absorption factor from
GIT.
Procedure:
Sodium benzoate is conjugated with glycine to form
hippuric acid, which is excreted in the urine. Sodium chloride is added to
decrease the solubility of hippuric acid and enhance its precipitation from
acidified urine. Hippuric acid is then isolated, dissolved and titrated with a
standard solution of alkali using phenolphthalein as an indicator.
Normal values:
Normal excretion of hippuric acid expressed as benzoic
acid
Oral = 3.0 –
3.5 g/4 hours
I.V. = 0.6 –
0.9 g/2 hours
Clinical significance:
Normal liver cells are important for the proper rate of
conjugation and excretion of a foreign substance when kidney function is
normal. Decreased rates are observed in liver cell impairment. In conditions
involving renal impairment, the results of the tests are inconclusive.
MISCELLANEOUS LIVER FUNCTION TESTS
1.
Cholesterol
The liver is concerned with the
metabolism of lipid especially cholesterol. Cholesterol is elevated in
obstructive jaundice and this increase parallels the increase bilirubin.
Changes in ratio of free to esterified cholesterol are seen in liver diseases.
When there is liver damage like infections or in viral infections, there is
fall of the cholesterol esters. Severe acute necrosis of the liver usually
shows a decrease of the total cholesterol and the cholesterol esters.
2.
Prothrombin time
This is reduced in liver diseases
particularly when damage of the hepatic cells due to the reduced production of
prothrombin. Decreased production of prothrombin may be bought about in two
ways:
a. In obstructive jaundice, the absence of
bile salts severely reduce the absorption of Vitamin K from the intestine.
b. Less production of prothrombin occur in
liver diseases with injury to the hepatic cells even in the presence of
adequate amount of vitamins.
These two conditions may be
distinguished by the Vitamin K tolerance test.
3.
Vitamin K Tolerance Test
a. Determine the prothrombin time on
several different to find the range or level for the patient.
b. Then give intramuscularly 76 mg Synkavit
for 4 consecutive days.
c. Determine the prothrombin time daily
just before the injection and on the day following the last injection
d. In normal individual, there is normal
prothrombin time and no change occurs following administration of Vitamin K.
e. In Vitamin K deficiency, the prothrombin
time is elevated and is decreased or return to normal following administration
of Vitamin K. In this condition, the
liver function is not seriosuly impaired.
4.
Fetoglobulins
Also known as alpha–fetoprotein which
has been noted in the serum of about two third of patients with primary
hepatocellular carcinoma. Fetoglobulin is present in fetal serum during the
development of the fetus, but falls to a low level and disappear completely by
the first week after life.
The protein is determined by agar or
cellulose acetate electrophoresis while the test is specific for carcinoma of
the liver, not all hepatocellular carcinoma give a positive test.
Fetoglobulin may also be present in the
serum of some patients with testicular tumor.
ENZYMES RELATED TO LIVER FUNCTION
A. Hepatocellular enzymes – used for the evaluation of hepatic
diseases reflecting active liver damage, both chronic and acute.
1. Alanine aminotransferase (ALT/SGOT)
2. Aspartate aminotransferase (AST/SGPT)
3. Gamma glutamyltransferase (GGT)
4. Alkaline phosphatase
5. Lactate dehydrogenase (LDH–5 and LDH–4)
6. Aldolase
B. Obstructive liver enzyme – used for the evaluation of hepatic
diseases reflecting hepatocellular injury from cholestasis or biliary
obstruction.
1. Alkaline phosphatase (ALP)
2. Gamma glutamyltransferase (GGT)
3. 5’–nucleotidase
4. Leucine aminopeptidase (LAP)
· Discussions of the above mentioned
enzymes can be seen on Lecture # 11: Enzymology.
DISEASES ASSOCIATED WITH LIVER
MALFUNCTION
I.
Jaundice
Jaundice, also known as Icterus is the most
common manifestation of liver diseases signifying hyperbilirubinemia,
characterized by the yellowish discoloration of the skin, mucous membranes and
sclera of the eyes. Conjugated bilirubin causes more jaundice than unconjugated
bilirubin because of its easier absorption into tissues and higher water
solubility. This form of bilirubin is
easily bound to elastic tissue and other tissues that have a high protein
content, making the yellow color particularly evident.
Classification of
Jaundice:
A. Pre–hepatic jaundice is caused by:
1. Excessive destruction of circulating
erythrocytes (hemolysis).
2. Ineffective erythropoeisis is a
pathologic process in which a very low proportion of red cells formed in the
bone marrow enters the circulation and those remaining in the bone marrow are
prematurely destroyed. An increase in the amount of bilirubin released from the
bone marrow results and is called early–labeled bilirubin since the bilirubin
has not been circulating within RBC for 120 days.
3. Increased turnover of non–hemoglobin
heme compounds in the liver and other organs.
4. Phagocytic breakdown of extravasated RBC
(hematoma)
B. Hepatic jaundice
1. Retention jaundice – cause by a defect in the transport of
unconjugated bilirubin into the hepatocyte.
2. Regurgitation jaundice – occur when hepatic cell is damaged or
defective or the excretion of products from hepatocyte is impaired.
C. Post–hepatic jaundice or obstructive
jaundice – is caused by
a blockage of the flow of bile from the liver.
D. Neonatal jaundice
This is a condition defined as having
total serum bilirubin levels above 15 mg/dl in few days after birth or
bilirubin levels persisting above 10 mg/dl for more than two weeks.
The enzymes necessary for metabolism and
conjugation are not present in sufficient concentrations at birth and do not function efficiently for a few
days afterward. These two conditions, as well as an increased rate of
absorption of unconjugated bilirubin from the infant’s intestinal tract, often
cause bilirubin levels to rise to 10 mg/dl before the liver can begin to clear
the excess bilirubin from the plasma.
Kernicterus is the deposition of unconjugated
bilirubin in the CNS that may cause severe neurologic damage. Interventions may
include the administration of phenobarbital to induce enzyme activity or
phototherapy with monochromatic blue light to cause the oxidation of bilirubin
to move soluble end products and enhance the renal excretion of bilirubin.
Expected laboratory results in Pre–hepatic, Hepatic and
Post–hepatic jaundice conditions:
Liver Function
Test Pre–hepatic Acute
Hepatocellular Chronic Post–hepatic
Jaundice
Jaundice Hepatocellular Jaundice
Total bilirubin N
to I I
I I Conjugated
bilirubin N
to I I
I
Unconjugated bilirubin I I
I I
Urine urobilinogen I I
I D
Urine bilirubin N I I I
Albumin N N
D N
Globulin N N
I N
Aminotransferase N I
I N
Alkaline phosphatase N N
N I
Lactate dehydrogenase I I
I N
BSP dye test N I
I I
Prothrombin time N N Prolonged N
II.
Congenital extrahepatic biliary atresia
Extrahepatic biliary atresia is an acquired defect
causing a serious and aggressive condition in which the extrahepatic bile ducts
become inflamed and increasingly non–functional. A total bilirubin level of 15
mg/dl is seen in this patients. Females are affected more often than males, and
it occurs in approximately 1 in 10,000 live births. Increasing jaundice and
hepatomegaly are seen within the first 2 to 3 weeks of life and affected
infants have diarrhea and steatorrhea from the lack of bile acids to aid in
digestion. The disorder results in an obstructive disease that leads to
cirrhotic liver failure and death within two years if not treated successfully.
The clinical and laboratory picture is similar to that of
any obstructive process. The key to prompt diagnosis and treatment is the
differentiation of extrahepatic biliary atresia
from prolonged neonatal jaundice, a true hepatic jaundice or other
correctable obstructive diseases. The hyperbilirubinemia is primarily of the
conjugated type in biliary atresia (more than 75%), whereas physiologic jaundice
results in an unconjugated bilirubinemia. Serum enzymes and lipoprotein X are
unreliable because these are all elevated in obstructive biliary disease. In
patients with physiologic jaundice, the administration of phenobarbital induces
liver enzyme activity and a rapid return to reference ranges for laboratory
results is seen. This is not the case with biliary atresia. A liver biopsy is a helpful tool in prompt diagnosis
and should not be delayed in those infants with a conjugated hyperbilirubinemia.
Surgery to alter the path of removal of bile from the
liver is the only treatment available for those patients with minimal blockage
of bile ducts. For most of those affected. Surgery is not indicated because it
does not correct the inherent defect. Orthotopic liver transplantation is the
only effective treatment for these patients. With recent advances in surgical
techniques and immunosuppression therapies, liver transplantation is
becoming the treatment of choice for
this condition.
III.
Hemolytic anemias
Hemolysis refers to premature erythrocyte
destruction and includes both ineffective erythropoeisis and increased lysis.
Liver function studies are helpful in the assessment of the severity of the
hemolytic process but should not be used for an initial diagnosis. Specific
laboratory testing to identify the source of the hemolysis is required. The
liver is functioning properly in these situations and only when the liver’s
ability to dispose of the excess bilirubin is exceeded will liver function
studies be abnormal.
IV.
Cirrhosis
Cirrhosis literally means a yellow–orange condition of
the liver. The architecture of the liver is permanently destroyed and this
condition is the end stage of several disease processes.
A. Wilson’s disease
B. Alpha–1–antitrypsin deficiency
C. Hemochromatosis
D. Primary Biliary cirrhosis
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