UNIVERSITY OF GUYANA,

COLLEGE OF MEDICAL SCIENCES

SCHOOL OF ALLIED HEALTH

CLINICAL
PATHOLOGY
GROUP A
Fat soluable vitamins
Lecture: Ms. Bibi-Ally Charles
Course Name: DMT-4106
 Group Members
NAME USI
Lloyd Moffatt 1028601
Ulanda Greene 1030225
Judea Daw 1028222
Alexus Michael 1032096
Tiffany Roberts 1014440
Satie Prittipaul 1029680
Kaibian Daly 1016802
Niron Ramdeen 1030327
Keneisha Ward 1024178
 OBJECTIVES

 To understand what are Fat soluable vitamins

 To learn about the sources and the importance of daily intake
 To learn about their structure, metabolism and deficiencies
 To learn about its clinical signifcants
 To understand the methodology of the Laboratory assessment
 INTRODUCTION
What are vitamins?
• Vitamins are a class of complex chemical molecules that the body
need in little amounts to stay healthy
❖Fat soluble
• These vitamins are soluble in fat-soluble organic solvents. They
coexist with fats in diet.
• The fat soluble vitamins are A,D,E,K.
 INTRODUCTION

Some characteristics of fat-soluble vitamins are :

●Adsorbed with dietary fat in micelles

●Excreted much more slowly

●Stored in adipose tissue & liver and hence they pose a greater risk of
toxicity when consumed in excess
VITAMIN A
 What is Vitamin A?
• Refers to fat soluble compounds such as
retinol, retinyl palmitate, and beta-carotene

• Can be obtained in two different forms,

known as preformed vitamin A and
provitamin A

• The preformed vitamin A is obtained from

animal sources whereas provitamin A is
obtained from plant based sources
COMMON USES OF VITAMIN A
DEFICIENCY IN VITAMIN A
• Vision
• Prolonged dietary deprivation
• cellular differentiation
• Decreased bioavailability of
• epithelial barrier function
provitamin A carotenoids
• immune function
• Interference with absorption,
storage, or transport of vitamin A
Chemical Structure
 Signs and symptoms of Excessive
vitamin A intake:
CLINICAL MANIFESTATION
Headache
Night Blindness Birth defects

Xerophthalmia  Bone thinning

Liver damage 
Respiratory Infections may occur
Skin irritation
Keratinization of epithelial cells Diarrhea and Nausea
Pain in the joints and bone
LABORATORY ASSESSMENT
Method : High-performance liquid chromatography
Principle
• The separation principle of HPLC is based on the distribution of the
analyte (sample) between a mobile phase (eluent) and a stationary phase
(packing material of the column).

• Depending on the chemical structure of the analyte, the molecules are

retarded (delay or held back)while passing the stationary phase
Principle
• After the analytes have left the column, a detection unit (such as a UV
detector) detects them. A data management system (computer software)
converts and records the signals, which are subsequently shown in a
chromatogram
Laboratory Assessment cont’d
• Sample - SST (Gold, marble or red) tube

• Test preparation - Fasting overnight (12-14 hours) (infants-draw prior to

next feeding)

 Reference range
• 0 to 30 d - Not established
• 1 m to 5 y - 14.4 − 42.6 ul/dL
• 6 to 11 y - 18.2 − 45.7 ul/dL
• 12 to 19 y - 18.8 − 54.9 ul/dL
• 20 to 39 y - 18.9 – 57.3 ul/dL
• 40 to 59 y- 20.1− 62.0 ul/dL
• >59 y - 22.0 − 69.5 ul/dL
VITAMIN D
 What is Vitamin D?
• Vitamin D is a fat-soluble vitamin that
facilitates the absorption of phosphate
and calcium in the intestine.

• It is essential for sustaining optimal

levels of calcium in the blood so bone
development, mineralization, and
remodeling to occurs.

• This important vitamin controls

metabolism and development of cells,
regulation of immune function and
inflammation reduction.
 Metabolism &
Synthesis of Vitamin D
Chemical Structure
Clinical Significance
• Vitamin D measurement is critical because it can be used to diagnose the
development and progression of metabolic disorders, neurological
illnesses, and cancer.

• It can also be used for persons who are not responding to supplemental
treatment with vitamin D.

• When there is a deficiency of vitamin D, it results in loss of bone density,

rickets, and other diseases. When there is too much vitamin D in the body,
it results in nausea, vomiting, weakness, problems with heart rhythm, etc.
Laboratory Assessment
Principle:
• Two principles: indirect immunochemical and direct chromatographic.

• Immunochemical techniques are used in routine testing because they are

simple.

• Chromatographic assays, on the other hand, are more precise and

dependable and are widely used; this uses high-performance liquid
chromatography (HPLC), which analyses the circulating amounts of 1,25-
hydroxy vitamin D in plasma/serum.
Laboratory Assessment
• HPLC technique is utilized to directly quantify vitamin D molecules in the
blood. It uses organic solvents to precipitate, extract, and separate vitamin D
metabolites.

• The different attraction strength of metabolites to the solid phase causes

chromatographic phase separation. Peaking occurs on the chromatogram
where it is measured by quantifying the amount of vitamin D present using a
UV detector
Laboratory Assessment cont’d
Required Sample:
Red top/purple top tube

Special test preparations:

Fasting specimen is recommended for 4 to 8 hours but not required

Materials:
• Serum calibrators (cholecalciferol)
• Controls (levels I, II, III)
• Pipette (400µl)
• Vortex mixer
• Centrifuge
• HPLC apparatus
• Test tubes
METODOLOGY

1. Pipette 400µl of serum (calibrator, control, or patient sample) into 2ml test
tubes.
2. Add 400µl each of the precipitation and extraction reagents to the tube.
3. Vortex mixed tubes for 10 seconds to obtain a flocculent precipitate and
centrifuged at 10,000 rpm for 5 minutes.
4. Discard the clear supernatant into a 10ml glass vial, capped, and place into
the auto sampler unit of the HPLC apparatus .
5. Results are displayed on the analyzer.
 Normal range
• Children up to 12 months old: 400 IU

• 1 to 70 years old: 600 IU

• Persons over 70 years old: 800 IU

VITAMIN E
 What is Vitamin E?
• Several forms, but alpha-tocopherol
is the only one used by the human
body
• Its main role is to act as an
antioxidant
• It also enhances immune function
and prevents clots from forming in
heart arteries.
Chemical Structure
Clinical Significance

• Evaluation of individuals with motor and sensory neuropathies

• Monitoring vitamin E status of premature infants requiring

oxygenation

• Evaluation of persons with intestinal malabsorption of lipids

Laboratory Assessment
Method : High-performance liquid chromatography
Principle
• The separation principle of HPLC is based on the distribution of the analyte
(sample) between a mobile phase (eluent) and a stationary phase (packing
material of the column).
• Depending on the chemical structure of the analyte, the molecules are retarded
(delay or held back)while passing the stationary phase
• After the analytes have left the column, a detection unit (such as a UV detector)
detects them. A data management system (computer software) converts and
records the signals, which are subsequently shown in a chromatogram
• Sample
Serum separating tube (SST in gold, marble or red top)

• Test preparation
Fasting overnight (12-14 hours) (infants-draw prior to next feed)
Method
1.Vortex mixed the serum sample (20 μl) with 80μl of 0.1 percent ascorbic acid and 100
μl of ethanol.
2. Extract the mixture twice with 1 ml of hexane
3.In a centrifugal vacuum concentrator, dry the extract
4.For HPLC analysis, dissolved the residue in 100μl of methanol.

REFERENCE VALUES:
• 0-17 years: 3.8-18.4 mg/L
• > or =18 years: 5.5-17.0 mg/L
VITAMIN K
What is Vitamin K?
● Vitamin K is a group of structurally
similar fat-soluble compounds found
in foods that share a quinone ring.

● They differ in length and degree of

saturation of the carbon tail and the
number of isoprene units in the side
chain.
2D Chemical Structure
3D Structure of vitamin K
 Importance of Vitamin K

Vitamin K promotes the generation of coagulation factors.

Coagulation factors are molecules that aid with blood clotting and
minimize excessive bleeding. It is also thought to contribute to the
prevention of bone loss.
Clinical Manifestation of Vitamin K
Deficiency
● Easy bruising
● Mucosal bleeding
● Oozing of blood from incision sites.
● Osteoporosis/Weak bones
● Excessive bleeding/Thin blood that doesn't clot
well
Prothrombin Time (PT) test

● The prothrombin time (PT) test

determines how long the blood clot
takes to develop in a patient's
blood.

Normal Range:
● 11-15 seconds
 Activated Partial Thromboplastin Time

● The APTT in contrast to the

Prothrombin Time, measures the
activity of the intrinsic and
common pathways of
coagulation
 Principle of Prothrombin Time test (PT)
The Prothrombin Time is a one-stage test based upon the time required for a fibrin

clot to form after the addition of Tissue Factor (TF - historically known as tissue

thromboplastin), phospholipid and calcium to de-calcified, platelet poor plasma.

The PT measures the activity of the so-called extrinsic and common pathways of

coagulation and therefore, is dependent on the functional activity of factors VII, X,

V, II (Prothrombin) and Fibrinogen.

Materials
● Gloves ● Tissue factor
● Disposable syringe ● Water bath
● Tourniquet ● Thermometer
● Test tube
● Calcium chloride
● Centrifuge
● Timer

Specimen : Blood
 Methodology
1. A venipuncture blood sample is taken off from the patient.

2. To avoid the clotting process from occurring before the test, the blood is decalcified

(by depositing it in a tube with oxalate or citrate ions).

3. After that, the blood sample is centrifuged.

4. The PT test is done by mixing the patient's plasma with a Tissue Factor (e.g., thromboplastin,

a protein derived from homogenized brain tissue) which initially converts prothrombin to

thrombin.
5. The mixture is then maintained for approximately 1-2 minutes in a warm water

bath at 37°C.

6. To counteract the sodium citrate and allow clotting to begin, calcium chloride is

added to the mixture.

7. The test is timed as from time the calcium chloride is added until the plasma

clots. This is known as the Prothrombin Time.

Interpretation of Results

Prolonged APTT + Prolonged PT indicates Vitamin K

deficiency

● Liver disease due to

-Malabsorption of Vitamin K and which leads to decreased gamma
carboxylation of the vitamin K dependent clotting factors.
Thank You
for
Watching
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