MEDT19 CLINICAL CHEMISTRY (LABORATORY)

LESSON 1: LIVER FUNCTION TEST

Learning objectives:

1. identify tests necessary in evaluating liver function;

2. determine the importance of liver function test in the diagnosis of hepatobiliary disorders;
3. interpret normal or abnormal results based on different methods used in the liver function evaluation.

INTRODUCTION:

Liver function tests, also called hepatic panel, are blood tests used to monitor liver function and damage. These tests provi de
insights into several aspects of liver health, notably: the liver’s ability to synthesize enzymes and proteins; the liver’s ability
to process bilirubin and secrete bile; and the extent of liver damage. Abnormal liver function test results do not always
indicate liver disease. Some abnormalities are transient; or they may result from different, non-hepatic causes.

ANATOMY OF THE LIVER

• largest and the most versatile organ in the body

• reddish-brown organ consists of two main lobes
• weigh from 1200-1500 grams in the normal adult

BIOCHEMICAL FUNCTIONS

• Major Synthetic Activity

o Production of plasma proteins
i. Synthesizes albumin and most of the alpha and beta globulin (except the immunoglobulins)
ii. All the blood clotting factors (except VIII)
iii. Synthesizes positive and negative acute-phase reactants and coagulation proteins.
o Metabolism and synthesis of carbohydrates
i. Metabolize carbohydrates; major player in maintaining stable glucose concentration performing
glycogenesis, glycogenolysis and gluconeogenesis.
o Metabolism of fat
o Major site of metabolic clearance of many other hormones
o Synthesizes enzymes which were found useful in the diagnosis of hepatobiliary disorders: AST, ALT, ALP, 5’NT
and GGT.
• Detoxification and Drug Metabolism
o Serves to protect the body from potentially injurious substances absorbed from the intestinal tract and toxic by-
products of metabolism.
o Ammonia (toxic by-product) is converted to urea in the liver.

• Excretory and Secretory Function

o Excretion of bile – bile acids or salts, pigments and cholesterol
o Excretion of bilirubin in the body
• Storage
o Storage site of all fat soluble and water-soluble vitamins
o Storage depot of glycogen
• Conjugation Function
o Involves in bilirubin metabolism.
o 200 – 300mg of bilirubin is produced daily in the healthy adult.

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Bilirubin Determination

• Bilirubin is derived from hemoglobin arising from the destruction of aged/old/senescent RBCs
✓ The heme portion will be converted into bile pigment – bilirubin (yellow pigment)
✓ During RBC destruction it undergo two process of hemolysis.
i. Intravascular hemolysis (10%) – occurs when hemoglobin is broken-down in blood, releasing free
hemoglobin. This free hemoglobin will bind in to hemopexin, haptoglobin and albumin in will be
phagocytized by the liver macrophage (haptoglobin: major free hemoglobin transport protein)
ii. Extravascular hemolysis (90%) – happens when old or damaged RBCs are being phagocytized in the
spleen or liver by a specific macrophage.

Bilirubin Metabolism:

RBC (will explode after 120 days)

Release free hgb (can be found in plasma)

Free hgb will form 3 fragments:

a. Globin – considered as protein portion of hgb; will return back to circulation

because it is essential in erythropoiesis
b. Iron – will be stored in tissue or bone marrow as “ferritin”; some will be converted
to “hemosiderin”; Iron is important in the development of erythroblast in BM.
c. Porphyrin ring – main precursor of bilirubin

hemoxygenase

Biliverdin (reduced form of bilirubin)

PRE-HEPATIC Bilirubin reductase

Bilirubin (B1/ unconjugated/indirect/non-soluble/non-polar bilirubin)

*needs to be attached to albumin for it to be transported to the

liver

Liver (where conjugation of bilirubin takes place)

UDPGT (Uridine Diphosphate Glucuronyl Transferase)

Bilirubin monoglucuronide (incomplete conjugated form of bilirubin)

HEPATIC
UDPGT (Uridine Diphosphate Glucuronyl Transferase)

Bilirubin diglucuronide (B2/ conjugated/ direct/ water-soluble bilirubin)

Bile (excreted by the liver in bile)

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Small intestine

Enterobacteriaceae – largest family of gram-negative

POST-HEPATIC bacteria/normal flora of the intestine; converts bilirubin into
urobilinogen thru the process called hydrolysis

Urobilinogen (good indicator whether your liver is conjugating or not)

Reabsorbed Oxidation

Reconjugated Stercobilinogen

Urobilin Stercobilin
(a type of urochrome responsible (a type of urochrome responsible
for the yellow color of urine) for the brown color of feces)

Key points:
✓ Presence of small amount of urobilinogen in the urine is normal.
✓ Presence of bilirubin in urine is abnormal.
✓ Total bilirubin is the one measured in the laboratory.

Clinical conditions:
1. Kernicterus – deposition of unconjugated bilirubin in the brain leading to mental retardation.
2. Jaundice – yellowish discoloration of the skin and mucus membrane of the eye.
✓ Patient has increased bilirubin in the blood
✓ If bilirubin is >2mg/dl - bilirubin goes out to soft tissue including sclera of the eye, skin and gums.
✓ If bilirubin is >20mg/dl – serum will appear dark yellow (icteric/icterus)

Three types of Jaundice:

1. Hemolytic jaundice – there is a rapid RBC destruction and cause hemolytic anemia
2. Obstructive jaundice – blockage of biliary passages; bilirubin leaks in the blood.
3. Hepatocellular jaundice – liver damage/ disease.

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METHODS OF LIVER FUNCTION EVALUATION:

Specimen requirements:
▪ Fasting serum specimen is recommended as the presence of lipemia will increase the measured bilirubin
concentration
▪ Non-hemolysed specimen, hemolysis causes false low results
▪ Sample must be protected from direct exposure to light (UV light) because bilirubin is photosensitive and breaks down
upon exposure to light. (During blood collection, tube must be covered with a carbon paper to avoid exposure to light)
▪ Sample should be measured 2-3 hours after collection.

1. Evelyn and Malloy Method

Principle: Van der Berg reaction is diazotization of bilirubin to produce azobilirubin.
• Diazo Reagent:
Diazo A = 0.1 % Sulfanilic Acid + HCl
Diazo B = 0.5 % Sodium Nitrite
Diazo blank = 1.5% HCl
• Direct Bilirubin
Bilirubin + Diazo Reagent → Azobilirubin (Pink to Purple) (Acidic)
• Total Bilirubin
Bilirubin + Methanol+ Diazo Reagent → Azobilirubin (Pink to Purple) (Acidic)
▪ Solubilizer/ Coupling Accelerator : Methanol
Indirect Bilirubin
Total Bilirubin – Direct Bilirubin = Indirect Bilirubin (Unconjugated)

2. Jendrassik and Grof Method

Principle: Van der Berg reaction is diazotization of bilirubin to produce azobilirubin.
• Buffer: Sodium Acetate
• Ascorbic Acid – stop the reaction
• Alkaline tartrate - to make the solution alkaline
• Direct Bilirubin
Bilirubin + Diazo Reagent + Ascorbic + Alkaline Tartrate → end-product (Blue) (Alkaline)
• Total Bilirubin
Bilirubin + Diazo Reagent + Caffeine–Benzoate + Ascorbic + Alkaline Tartrate →(Blue) end-product
▪ Coupling Accelerator : Caffeine – Benzoate ( preferred over methanol because methanol
promotes protein precipitation and increases turbidity)
• Indirect Bilirubin
Total Bilirubin – Direct Bilirubin = Indirect Bilirubin (Unconjugated)

Reference Range:
Direct Bilirubin: 0 - 0.2mg/dl (0-3 umol/L)
Indirect Bilirubin: 0.2 – 0.8mg/dl (3 – 14 umol/L)
Total Bilirubin: 0.2 – 1.0mg/dl (3 – 17 umol/L)

Key Points:
✓ Jendrassik and Grof method is the reference method for bilirubin determination because it is the most sensitive, most
popular in automation and most widely used.

3. Direct Spectrophotometric method

• Not practical for adult but has value in the evaluation of jaundice in newborns
• Only valid for newborns whose serum does not contain lipochromes.
• The absorbance of hemoglobin at 455nm is corrected by subtracting the absorbance at 575nm
Specimen requirements:
✓ Serum is collected and stored with the same precautions for adult specimens.
✓ Specimen can also be obtained through skin puncture technique since newborn can have difficulties in blood
collection through venipuncture.
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Sources of error:
✓ Error will be introduced if the buffer is turbid
✓ Because this method depends on the extinction coefficient of bilirubin, all volumes must be accurate and cuvets must
be flat-surfaced, with a path length of exactly 1 cm.
✓ This method is relatively insensitive to hemolysis, which is often present in specimens obtained from infants, due to
difficulty in skin puncture technique.

Reference Range:

Infants Premature, total Full term, total

24 hours 1 – 6 mg/dl 2 – 6 mg/dl
48 hours 6 – 8 mg/dl 6 – 7 mg/dl
3 – 5 days 10 – 12 mg/dl 4 – 6 mg/dl

Key Points:
✓ Bilirubin conversion factor: 17.1

4. Icterus Index
• a measure of the degree of icterus (yellowishness) of plasma or serum specimen in cases of jaundice due to
hemobilirubin and cholebilirubin.
Test:
▪ Involves diluting the serum with saline (or sodium citrate solution) until it visually matches the color of a 0.01%
potassium dichromate standard solution.
▪ The number of times the serum must be diluted is called the Icterus Index.
▪ Units: Absorbance of specimen/absorbance of standard x 10
▪ Reference value: 3 – 8 icterus index
Methods:
▪ Muellengracht – 0.85% saline is the diluent
▪ Newberger – sodium citrate is the diluent
Sources of error:
o Substances in the serum other than bilirubin, such as carotene, xanthophylls, and hemoglobin may also contribute
to the icterus index, limiting its usefulness thus, this test is now obsolete.

5. Urine Bilirubin Tests

5.1 Foam Test:
About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also
obtained with proteins and highly concentrated urine. In normal urine, foam is white
5.2 Fouchets test:
This is a simple and sensitive test.
i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of
sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex).
ii. Filter to obtain the precipitate on a filter paper.
iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25
grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml).
iv. Immediate development of blue-green color around the drop indicates presence of bilirubin

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5.3 Ictotest Tablet Test:

Diazotization of urine bilirubin under acid pH produce a blue to purple azobilirubin; the reagent contains SSA and p-
nitrobenzene diazonium p-toluene sulfonate; confirmatory test.

6. Urobilinogen in Urine and Feces

6.1 Urine Urobilinogen
o By (urine) dipstick which is based on the reaction with p-dimethylaminobenzaldehyde (Ehrlich’s reagent)
Specimen:
✓ Fresh 2-hr urine which should be kept cool and protected from light
Principle:
✓ urobilinogen in urine + Ehrlich’s reagent red color (spectrophotometer)
✓ Ascorbic acid is added as reducing agent to maintain urobilinogen in the reduced state.
✓ The use saturated sodium acetate stops the reaction and minimizes the combination of other chromogens
with the Ehrlich’s reagent.

Sources of error:

✓ Fresh urine is necessary, and the test must be performed without delay to prevent oxidation of urobilinogen to
urobilin.
✓ Spectrophotometric readings should be made within 5 minutes after color production because urobilinogen-
aldehyde color slowly decreases in intensity.

Reference Range:

✓ 0.1 – 1.0 Ehrlich units/ 2 hours

✓ 0.5 – 4.0 Ehrlich units/day

6.2 Fecal UrobilinogenVisual inspection of the feces usually suffices to detect decreased urobilinogen because the stool
becomes pale or clay colored.
Specimen:
o Aqueous extract of fresh feces

Principle:

o Urobilin alkaline ferrous hydroxide urobilinogen + Ehrlich reagent red color

Reference Range:

• 75 to 275 Ehrlich units per 100 grams of fresh feces

• 75 to 400 Ehrlich units per 24-hour specimen

7. Serum Bile Acids

o Complex methods are required for the analysis of bile acids in serum:
a. Extraction with organic solvents e. ultraviolet light absorption
b. Partition chromatography f. Flourescence
c. Gas chromatography- mass spectroscopy g. radioimmunoassay
d. Spectrophotometry h. enzyme immunoassay

Specimen:
o Postprandial serum sample allows a more sensitive distinction between normal and abnormal values than
testing a fasting sample, because the usual increased in secretion is stimulated by eating.
Key Points:
✓ Increases serum bile acids = liver disease
✓ Ratio of trihydroxy to dihydroxy bile acids in serum – differentiates patients with obstructive jaundice from
those with hepatocellular jaundice

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✓ Ratio of cholic and chenodeoxycholic acid in serum – aids in the diagnosis of primary biliary cirrhosis and
extrahepatic cholestasis.
✓ High Ratio – Biliary Obstruction
✓ Low Ratio – Primary Biliary Cirrhosis

8. Liver Enzymes
Differentiate hepatocellular (functional) from obstructive (mechanical) disease.
Specimen:
✓ Serum is the specimen of choice for most of the liver enzyme test. Blood is usually collected together with
other test that uses serum as its sample.
8.1 AST/ ALT (Aminotransferases)
• damaged or necrotic hepatocytes
• High concentration-drug hepatotoxicity
8.2 ALP (Phosphatase)
o Used most often in clinical diagnosis of bone and liver disease.
o Most striking elevations (up to 20x) occur in extrahepatic biliary obstruction.
8.3 5’-nucleotidase
• Used clinically to determine whether ALP elevation is caused by liver or bone disease
• Increased ALP, increased 5’-nucleotidase = LIVER
• Increased ALP, normal to slightly elevated 5’-nucleotidase = BONE
8.4 GGT
• Elevated in the serum of almost all patients with hepatobiliary disorders
• Highest level are seen in biliary obstruction (cholestasis)
• chronic alcohol and drug ingestion
• level returns to normal after 3- 6 weeks of absentation from alcohol, making it a useful test for compliance in
alcohol reduction programs.
8.5 LD
• LD 4 and 5 (Skeletal Muscle and Liver)
▪ Acute Hepatitis and Cirrhosis

9. Ammonia
Product of amino acid deamination which is toxic to the body.
Specimen:
✓ Hemolyzed specimen should not be used
✓ Plasma (heparin/EDTA-Na2) kept on ice as much as possible in order to minimize formation of NH 3
✓ Specimen should be removed promptly from contact with red cells and must be analyzed ASAP.
✓ Refrigerated if not performed immediately; freeze if delayed beyond 24 hours
✓ Avoid using cleaning solution or hand cream that contain ammonia
✓ Avoid tourniquet, fist clenching: Arterial blood collection is preferred.
✓ Avoid exercise or any strenuous activity, and smoking prior to testing.

Methods:
1. Berthelot Reaction:

NH3 + Phenol + Hypochlorite Indophenol Blue

2. Enzymatic Assay:
✓ Glutamate Dehydrogenase (GD)
✓ Decrease in absorbance at 340

NH4 + α- ketoglutaric acid + NADH GD

Glutamic Acid + NAD+

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Sources of Error:
1. Glucose levels over 600 mg/dl(33.3 mmol/L) decreases the ammonia result by 8 – 40 umol/L
2. High BUN values (14.3 mmol/L) may increase values of ammonia by 14 umol/L.

Laboratory Analysis of Ammonia:

1. For the diagnosis of hepatic coma

2. For the diagnosis of Reye’s syndrome

TEST MEASURING HEPATIC SYNTHETIC ACTIVITY

A. TPAG – Cirrhosis (Decrease)

B. Albumin
C. A/G ratio
D. PT/ PTT
E. Gamma globulins
E.1. IgG and IgM
• Chronic active hepatitis
E.2. IgM
• Biliary cirrhosis
E.3. IgA
• Alcoholic Cirrhosis

AUTOIMMUNE MARKERS

A. Anti-mitochondrial Antibody
A.1. Primary Biliary Cirrhosis (PBC)
A.2. Method: ELISA
▪ AMA with Anti-M2 specificity (AMA-M2) is 100% specific for PBC

B. Anti-neutrophil cytoplasmic Antibodies (ANCA)

B.1. Primary Sclerosing Cholangitis
▪ Autoimmune disease associated with destruction of extrahepatic and intrahepatic bile ducts

C. Anti-nuclear Antibody and Anti-smooth muscle Antibody

• greater than 1:80 titer
• Type 1 Autoimmune Hepatitis

D. Antibodies to Liver-Kidney Microsomal Antigens

• Type 2 Autoimmune Hepatitis

OTHER TESTS

A. Hepatitis Profile

B. MELD Score (Model for End Stage Renal Disease)

o Bilirubin, INR (PT), Serum Creatinine
o Probability of survival of patients with liver disease
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▪ TIPS – Transjugular Intrahepatic Post-systemic Shunt

▪ > 24 MELD Score = TIPS not recommended

PGA Index
• Prothrombin Time (PT) - Prolonged
• Gamma glutamyl transferase (GGT) - Elevated
• Apo A1
▪ Increased – Alcoholic liver Hepatitis /Liver Injury
▪ Decreased - Cirrhosis

A1 Anti-trypsin, Ceruloplasmin
• Excretory function of the liver
• Ceruloplasmin – Removed by the liver
• AAT- hereditary deficiency; associated with early cirrhosis
Vitamin K Response Test
o Supplement – Vit. K
o Normal 2nd Test = Vit. K deficiency

Hippuric Acid Synthesis Test

• Assess the detoxification function of the liver
• Normal liver = benzoate is detoxified into hippuric acid
• Normal Result: Hippuric Acid in the Urine

References:

Bishop, Fody, and Schoeff. Clinical Chemistry Principles, Techniques, Correlations, Seventh Edition. 2013.

McPherson, Richard A. and Pincus, Matthew. Henry’s Clinical Diagnosis and Management by Laboratory Methods.
23rd Edition. Philadelphia: Elsevier Inc. 2017.

Burtis, Ashwood, Bruns. Tietz Fundamentals of Clinical Chemistry 6th edition. 2008

Anderson and Cockayne. Clinical Chemistry Concepts and Applications. 2003.

https://www.bioscience.com.pk/topics/pathology/clinical-pathology/item/823-test-for-detection-of-bilirubin-in-urine

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