Beruflich Dokumente
Kultur Dokumente
Lecture Outline
Introduction
Classification
Vitamin A
B complex
Vitamin C
Vitamin D
Vitamin E
Vitamin K
Vitamins
Initially thought to be amines when first discovered
Vitamines - substance that is essential for life and
yet needed only in minute amounts
Vitamins now known to be organic compounds, not
necessarily amines
Essential for growth and development
Dietary intake vital – body cannot synthesize them
all, or in sufficient amounts to fulfill body
requirements
Normal mixed diet ⇒⇒ adequate vitamins
Vitamins - Analysis
Deficiency vs. overdose
Deficiency
Inborn error of metabolism, unusual restriction of
dietary intake (food fad, poverty)
Complication resulting from other disease affecting
absorption of food
Use of certain drugs
Overdose
Excessive use
Medication
1.Water soluble
Vitamin C (ascorbate)
B complex
2.Fat soluble
A (retinol)
D (calciferol)
K (2-methyl-1,4,naphthoquinone)
E (α-tocopherol)
Vitamin A
Retinol and 3-dehydroretinol
Precursors of vitamin A = carotenes, found in yellow
and green parts of plants; especially abundant in
carrots
Active vitamin A formed by hydrolysis of β-carotene
in intestinal mucosa
Retinol transport: bound to α-globulin retinol-binding
protein
Stored in animal tissues, particularly liver
Vitamin A
Hypervitaminosis A
Vitamin A in large doses is toxic
Overdosage commonly due to excessive vitamin
use without medical advice
Acute poisoning/intoxication – reported in Arctic
regions as a result of consuming polar bear liver
Chronic hypervitaminosis A ⇒ fatigue, insomnia,
bone pain, hair loss, desquamation and
discolouration of skin
Vitamin A
Quantitation
Colorimetric method
Semi-quantitative
Chromogenic reagent reacts with retinol to produce a
blue colour → intensity is proportional to amount of
retinol in sample
Measure a blue colour against a set of known
standards
Blue chromophore is very transient, unstable
Necessitates fast and skillful personnel
Gaining popularity
Normal and reverse phase column and UV detector at
280 nm
Hexane used to separate retinol from other
substances which absorb radiant energy at equal or
similar wavelengths to retinol
Retinol detected using spectrophotometric or
flourometric methods
Rapid, specific, high resolution
High cost of instrument
Highly trained personnel required
Vitamin B complex
Thiamine (B1)
Riboflavin (B2)
Nicotinamide (niacin)
Pyridoxine (B )
6
Biotin
Pantothenic acid
Vitamin B complex
Fluorometric methods
Method of choice is thiochrome procedure
Treatment of thiamine with an oxidizing agent
(ferricyanide or hydrogen peroxide) to form a
flourescent compound (thiochrome)
Intensity is proportional to the thiamine
concentration
Riboflavine
Source: diet
Function:
Flavine mononucleotide (FMN)
Flavine adenine dinucleotide (FAD)
FMN and FAD = reversible electron carriers in
biological oxidation systems
Deficiency = ariboflavinosis
Ariboflavinosis
Illustration from
‘Disorders of malnutrition’
http://www.fao.org/DOCREP/W0073e/w0073e05.htm#P2916_330082
Riboflavine quantitation
Microbiological
Lactobacillus casei
Growth of this riboflavine organism correlates with the
amount of vitamin in the sample
Growth response measured by measuring turbidity
Riboflavine quantitation
Nutritional status
Urinary excretion:
Urinary determination from fasting, timed or 24 hour
samples
Measured by HPLC separation, fluorometric
detection
Load return test
Measurement of erythrocyte riboflavin
Determination of lysed erythrocyte FAD
Riboflavine is cofactor for glutathione reductase
Measure glutathione reductase activity from a small
amount of heparinized venous blood
Low erythrocyte activity of this enzyme ⇒
riboflavine deficiency
Riboflavine quantitation
N(1)-methyl-3-carboxamide-6-pyridone
3 forms
Pyridoxine (pyridoxol)
Pyridoxal – aldehyde
Pyridoxamine – amine
Etc.
Vitamin B6
HPLC
Deficiency:
Common in intestinal malabsorption syndromes,
e.g. ‘contaminated bowel’ syndrome
During pregnancy and lactation
Microbiological assay
Streptococcus faetalis, Lactobacillus casei
Liberate free biotin by proteolytic digestion
Other methods:
Competitive protein binding
Microbiologically:
Lactobacillus leichmannii
Pantothenic acid
Ascorbate
Water soluble vitamin
Found in fruits and vegetables
Cannot be synthesized by the body
Easily and irreversibly oxidized and loses its
biological activity in presence of oxygen, catalysed
by heat
Vitamin C
Functions:
Hydrogen carrier
Wound healing
-20°C
Whole blood
Leukocytes
Leukocyte assay immediately
Urine
Vitamin D
Fat-soluble
Manufactured by body on exposure to sun
Derived from:
Cholecalciferol (vitamin D )
3
Free-ionised hypercalcaemia
3 types:
Vitamin D
25-hydroxy vitamin D
1,25-dihydroxy vitamin D
Increase in polarity with increase in OH group
Can be used to separate the 3 types
chromatographically using alumina and silica
columns
Vitamin D quantitation
Easier to assay
Vitamin D quantitation
Extraction
Ethanol, methanol , diethylether and acetonitrile
(methyl cyanide)
Deproteinisation or denaturation of DBP
Purification
Column chromatography to separate the
metabolites from each other and from lipids and
other interfering substances
Vitamin D quantitation
Quantitation of 25(OH)D
UV absorption
large sample
RIA
Popular method
Antiserum reacts on equimolar basis with both D2
and D3
α-tocopherol
Soluble in fat solvents but not in water
Sufficient intake with a healthy diet
Deficiency rare because of easy availability in
food
Excess can be harmful
Known as a potent antioxidant
Vitamin E
Solvent extraction
Chromatography
2D reverse-phase paper, thin layer, column
chromatography,
Gas chromatography,
Chemical methods
Based on redox reaction
∀ ∴ deficiency of Vitamin K
⇒ predispose to bleeding
Vitamin K
Chromatographic separation
Quantitation
Vitamin K quantitation
Prothrombin time
Add tissue thromboplastin to recalcified
plasma and measure the time required for
clot formation
Vitamin K is necessary for synthesis of
plasma clotting factors
Vitamin K deficiency
Dr Ralph Leischner
http://www.lumen.luc.edu/lumen/meded/mech/cases/case11/list.htm
The END