Outline

• Liver function testing including:

 Bilirubin and bilirubin metabolism and correlation with

prehepatic, hepatic and posthepatic disorders.

 Proteins

 Liver enzymes

• Validation of results with quality control according to SOPs.

2
 Liver functions Testing contd.

 Proteins
 Biosynthetic Function of liver
• Total protein
• The total protein consists of albumin and globulin.

• Proteins are important parts of all cells and tissues.

• Albumin helps prevent fluid from leaking out of blood vessels

while globulins are an important part of the immune system.

•
• Albumin (not liver specific but does mirror
long-term hepatic synthetic function)

• Prealbumin

• Retinol binding protein

• Alpha and beta globulins

 Liver functions Testing contd..
• Alpha and beta globulins

• Alpha globulin
– Alpha-1-antitrypsin
– Ceruloplasmin
– α-fetoprotein

• Beta globulin
– Transferrin
– Sex hormone binding globulin
 Liver functions Testing contd.
 Proteins contd.
 Biosynthetic Function of liver contd.

• Coagulation factors

• Most factors are produced in the liver

• Factors II, VII, IX, and X are vitamin-K dependent

(factors require adequate quantities of vitamin K for
 Liver functions Testing contd.
 Enzymes
 Transaminases

 Markers of liver Transformational function

• Tests to Assess Liver Disease (Hepatocellular Damage)

• AST found in numerous tissues, including the liver,

cardiac muscle, skeletal muscle, kidneys, brain, and
pancreas
 Liver functions Testing contd..
• Transformation function of the liver/enzyme markers
contd.

• ALT found primarily in the liver

• AST and ALT: They are an excellent marker of hepatocellular

injury.

• They participate in gluconeogenesis by catalysing the

transfer of amino groups from aspartic acid or alanine to
ketoglutaric acid to produce oxaloacetic acid and pyruvic
acid respectively.
 Liver functions Testing contd.
Considered most specific laboratory test for liver injury.

Release of enzymes into the blood occurs when liver-cell

membrane is damaged.

– Limitations of AST and ALT as liver function markers

– Sensitive indicator of liver-cell injury; however, may not
be elevated in 10-15% of patients with chronic disease.

-Poor correlation exists between degree of liver damage and level

of aminotransferases.

-Level of aminotransferases does not provide prognosis for liver

necrosis or permanent damage
 Liver functions Testing contd.
• Biochemical tests of liver function contd.

• Patterns of elevation may suggest where abnormality exists:

– AST/ALT elevations – hepatocellular damage

– ALP, bilirubin elevations – cholestasis

• Elevated ALT, AST, gamma glutamyl transferase (GGT), and 5’

nucleotidase (5’ NT) – combined elevation highly suggestive of
liver disease.
 Liver functions Testing contd.
• Biochemical tests of liver function contd.

• Patterns of elevation may suggest where abnormality exists:

– AST/ALT elevations – hepatocellular damage

– ALP, bilirubin elevations – cholestasis

• Elevated ALT, AST, gamma glutamyl transferase (GGT), and 5’

nucleotidase (5’ NT) – combined elevation highly suggestive of
liver disease.
 Liver functions Testing contd.
– Striking elevation (<20x URL)

• Acute hepatitis

• Toxin/drug-induced hepatitis

• Ischemic damage to the liver

– AST/ALT ratios
• Normal – 0.8-1.0
 Liver functions Testing contd..
• Enzyme matkers contd.
• Cholestasis
• ALP isoenzymes include liver, bone, placental, and intestinal forms of ALP.

• Usually increased during periods of growth (eg, children and teenagers)

and during pregnancy.

• GGT very sensitive indicator of liver injury (even minor injury).

• Clinical value – determine origin of elevated ALP.

• 5’ NT very sensitive and specific for hepatobiliary disease.

• Clinical value – determine origin of elevated ALP.

 Liver functions Testing contd..
• ALP contd.
• ALP Up to 3-fold elevation may be seen in liver disease – not specific
for cholestasis.

• Multiple isoenzymes – liver, bone, intestinal, placental elevation of

ALP exclusive of other enzymes may indicate bone disease.
•
– ALP isoenzyme testing – differentiates between bone and liver as
source of ALP elevation.

– GGT/5’ NT – distinguish elevated ALP as liver related

• Elevated GGT/5’ NT – liver
• Normal GGT/5’ NT – bone
• Protein concentration determination and
Validation of results with quality control
according to SOPs.
• Total protein
• Principle:
• Protein in plasma or serum sample forms a blue coloured
complex when treated with cupric ions in alkaline solution.

• The intensity of the blue colour is proportional to the protein

concentration.

• This was quantified colorimetrically using direct Biuret method

(Friedman et al., 2003).

• Sample/ standard/ control are added to test tubes containing

total protein reagent which contains 6.0mmol/l potassium
iodide, 21mmol/l potassium sodium tartarate, 6.0 mmol/l
coppersulphate and 58mmol/l sodium hydroxide as shown in the
 Assay procedure for total protein

Reagents Blank Sample Standard Control

Total protein reagent 1000µl 1000µl 1000µl 1000µl

Distilled water 20µl - - -

Sample - 20µl - -

Standard (6g/dl) - - 20µl -

Control - - - 20µl

They were mixed and incubated for 10 minutes at 37oC and absorbance
read at 546nm using the reagent blank to zero the instrument.
• Calculation:
• Concentration of total protein (g/dl)=
• Absorbance of sample X concentration of
standard (6)
• _____________________________________
• Absorbance of standard
•
• Albumin concentration determination and
Validation of results with quality control
according to SOPs.
• Albumin
• Bromocresol green method was used to colorimetrically
quantify this (Friedman et al., 2003).

• Principle: The measurement of serum albumin is based on its

quantitative binding to the indicator 3, 3', 5, 5'-tetrabromo-
cresol sulphonapthalein (bromocresol green, BCG).

• The albumin-BCG complex absorbs maximally at 578 nm, the

absorbance being directly proportional to the albumin
concentration in the sample.

• Sample/ standard/control are added to test tubes containing

albumin reagent which contains: 75mmo/l succinate buffer (pH
4.2) and 0.14g/l bromocresol green as shown in the assay
 Table: Assay procedure for albumin

Reagents Blank Sample Standard Control

Albumin reagent 1000µl 1000µl 1000µl 1000µl

Distilled water 10µl - - -

Sample - 10µl - -

Standard (3g/dl) - - 10µl -

Control - - - 10µl

Tubes were mixed and incubated at room temperature for 1 minute and absorbance read
at 630nm against the reagent blank.
• Calculation:
• Concentration of albumin (g/dl)=
• Absorbance of sample X concentration of
standard (3)
• _____________________________________
• Absorbance of standard
• Enzymes activities determination and
Validation of results with quality control
according to SOPs.
• Aspartate aminotransferase (AST) or Serum glutamate oxaloacetate
transaminase (SGOT):
• This is quantified colorimetrically using L-aspartate and alpha-
ketoglutarate (Friedman et al., 2003).

• Principle: Kinetic determination of SGOT activity is by the following

reaction.
• L-Aspartate + α-ketoglutarate----AST----→oxaloacetate + L-Glutamate
• Oxaloacetate + NADH + H+---MDH--→L-Malate + NAD+
• 100µl of sample/ control is added to tubes containing 1000µl SGOT
working reagent which contains: 88mmol/ Tris Buffer (pH 7.8),
260mmol/l L-aspartate, ≥ 600 U/L malate dehydrogenase, ≥ lactate
dehydrogensae, 0.20mmol/l NADH and 12mmol/l alpha-
ketogluterate.

• The tubes are properly mixed and change in absorbance per minute
read at 340nm over 3 minutes using the kinetic mode and water to
• Calculation:
• Activity of SGOT U/L =
• Absorbance of test X molecular weight of SGOT X
total volume of reaction mixture
• ________________________________________
• Reaction time X extinction coefficient of SGOT at
340nm X volume of sample
•
• SGOT activity (U/L) =∆absorbance/minute X 1768
• Alanine aminotransferase (ALT) or serum glutamate pyruvate
transaminase (SGPT):
• This is quantified colorimetrically using L-alanine and alpha
ketoglutarate (Friedman et al., 2003).

• Principle:
• Kinetic determination of SGPT activity is based on the following
reaction:
• L-Alanine + α-Ketoglutarate----ALT---→ L-glutamate + Pyruvate
• Pyruvate + NADH + H+---LDH----→L-Lactate + NAD+

• 100µl of sample or control was added to tubes containing 1000µl of

SGPT working reagent which contains: 110mmol/Tris buffer pH 7.5,
550mmol/l L-alanine, ≥200U/L lactate dehydrogenase, 0.20mmol/l
NADH and 16mmol/l alpha-ketogluterate.

• They are mixed properly and change in absorbance per minute read
• Calculation:
• Activity of SGPT (U/L) =
• Absorbance of test X molecular weight of SGPT X
total volume of reaction mixture
• ________________________________________
• Reaction time X extinction coefficient of SGPT at
340nm X volume of sample
• Activity of SGPT U/L =∆absorbance/minute X 1768
• Alkaline phosphatase (ALP)
• This is quantified colorimetrically using p-nitrophenol phosphate
(Friedman et al., 2003).

• Principle:
• The kinetic determination of ALP activity is based on the following
reactions.
• P-Nitrophenyl phosphate + H2O---ALP----→p-Nitrophenol + Inorganic
phosphate

• 20ul of sample or control was added to tube containing 1000µl of ALP

reagent which contains: 125mmol/l diethanolamine buffer pH 10.2,
magnesium chloride- 0.625mmol/l and 50mmol/l P-nitrophenyl
phosphate.

• They are mixed and change in absorbance per minute read over
3minutes using distilled water to zero the instrument at the kinetic
• Calculation:
• Activity of ALP (U/L)=
• Absorbance of test X molecular weight of ALP X
total volume of reaction mixture
• ________________________________________
• Reaction time X extinction coefficient of ALP at
405nm X volume of sample
•
• Activity of ALP U/L = ∆absorbance/minute X 2750
• Validation Validation of results with quality
control according to SOPs.
• Controls are included for validation of results.
• Intra-laboratory and inter-laboratory quality
controls and assessment are done for routine
and periodic validation of results respectively
• Food for the day:

• Eph 1:7-8
• 7 In him we have redemption through his
blood, the forgiveness of sins, in accordance
with the riches of God's grace 8 that he
lavished on us with all wisdom and
understanding