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Lecture Outline
 Introduction

 Classification

 Vitamin A
 B complex

 Vitamin C

 Vitamin D

 Vitamin E

 Vitamin K
 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
 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

 Small concentration- micro/milligram

Vitamins - Classification
 On the basis of solubility
 Two groups:

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
 Active vitamin A formed by hydrolysis of β-carotene
in intestinal mucosa
 Retinol transport: bound to α-globulin retinol-binding
 Stored in animal tissues, particularly liver
Vitamin A

 Insoluble in water but soluble in organic solvents and

mineral oil
 Functions:
 Essential for normal mucopolysaccharide synthesis,
mucus secretion
 Rhodopsin:
Rhodopsin retinal pigment necessary for scotopic
vision – consists of protein opsin + Vitamin A
 Rhodopsin is destroyed in bright light

 Partial regeneration in the dark

 Vitamin A vital to maintain retinal levels

Vitamin A
 One of the most unstable micronutrients
 Sensitive to air, light, moisture, heat and acid
 Vitamin A absorption is recommended for
assessment of intestinal absorption
 Absorbance peaks:
 325 nm for retinol
 351 nm for 3-dehydroretinol
 Decreased levels seen in chronic infection,
fever, hepatic disease, and many disorders that
involve lipid metabolism
Vitamin A

 Prolonged deficiency of vitamin A very rare in

affluent communities; relatively common in poorer
communities – common cause of blindness
 Clinical effects of vitamin A deficiency:
 Night blindness due to rhodopsin deficiency

 Dry, squamous metaplasia of epithelial surfaces;

follicular hyperkeratosis; dry and wrinkled
conjunctiva and cornea – due to deficient mucus
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
 Blue chromophore is very transient, unstable
 Necessitates fast and skillful personnel

 This assay method is inexpensive, does not need

sophisticated equipment, thus used in many countries
Neeld-Pearson Procedure
 Collect fasting blood specimen
 Specimen must be free from haemolysis and
protected from light – method is sensitive to light
 Separate serum immediately
 Can be frozen for up to 2 weeks

 Extract vitamin A and β-carotene with petroleum ether

 Read absorbance at 450 nm ⇒ amount of β-carotene
 Evaporate and resuspend with triflouroacetic acid
 Read at 620 nm
 Correct reading for β-carotene

 Tedious, imprecise, non-specific

Spectrophotometric method

 The sample is irradiated with UV light and its

absorbance is measured
 The absorbance is proportional to vitamin A
content in the sample
 Can be used to determine vitamin A in fortified
HPLC Method

 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 )

 Folate / folic acid (pteroylglutamate)

 B complex (cobalamins)

 Biotin
 Pantothenic acid
Vitamin B complex

 Many synthesized by colonic bacteria

 Thiamine, folate, vitamin B12 ⇒ actively absorbed
from intestinal tract;
 The rest diffuse passively through intestinal
mucosal wall
 Most act as enzyme cofactors
 Clinical deficiency is rare in affluent communities

 Component of thiamine pyrophosphate:

essential cofactor for decarboxylation –
conversion of pyruvate → acetyl CoA
 Cannot be synthesized by
 Dietary intake: adequate
amounts in normal diet

 Deficiency can seen in alcoholism, anorexia nervosa

 Deficiency ⇒ pyruvate metabolism disrupted →
pyruvate ↑↑ accumulation in blood
 Beri-beri:
 Anorexia, emaciation, neurologic lesions, cardiac
 Occurs mainly in countries where staple diet is
polished rice
 May be aggravated by high carbohydrate diet ≈
dietary carbohydrate ↑ glycolysis ↑ pyruvate ↑
Illustration from
Thiamine quantitation
 Microbiological method
 Chemical conversion
 HPLC with UV detection
 Biochemical tests
 Based on functional level of TPP

 Reflect immediate nutritional status

 e.g. measure ratio of lactic to pyruvic acid in blood

after administration of glucose
 Other metabolites that increase due to deficiency
are 2-oxoglutarate, glyoxylate
 Most useful for assessing thiamine status is
measurement of whole blood or erythrocyte
Thiamine quantitation

 Whole blood or erythrocyte transketolase

 The enzyme catalyze 2 reactions in the pentose
phosphate pathway
 Look for disappearance of one of the substrates
 Xylulose 5-phosphate + ribose 5-phosphate →
sedoheptulose-7-phosphate + glyceraldehyde-3-
 Xylulose-5-phosphate + erythrose-4-phosphate →
fructose -6-phosphate + glyceraldehyde-3-phosphate
Thiamine quantitation

 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
 Source: diet
 Function:
 Flavine mononucleotide (FMN)
 Flavine adenine dinucleotide (FAD)
 FMN and FAD = reversible electron carriers in
biological oxidation systems
 Deficiency = ariboflavinosis

Illustration from
‘Disorders of malnutrition’
Riboflavine quantitation

 Many types of tests available

 Flourometric
 Microbiological
 Binding
 Riboflavin with egg white RBP
 FMN with apoflavodoxin
 With apoproteins for D-amino acid oxidase or
glucose oxidase
 Measuring characteristic yellowish green
riboflavine fluorescence

 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
 Measured by HPLC separation, fluorometric
 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

 Erythrocyte glutathione reductase activity

 Apoenzyme + FAD → HOLOenzyme

 GSSG+ NADPH + H+ → 2G-SH + NADP+

 Determine NADP formed spectrophotometrically

Nicotinamide (Niacin) B3

 Nicotinamide formed in the body from nicotinic acid

 Humans can synthesize some nicotinic acid from
 Both dietary and endogenous sources necessary to
provide enough nicotinamide for normal metabolism
Niacin B3
 Nicotinamide = active constituent of
 NAD+ - nicotinamide adenine dinucleotide

 NADP+ - nicotinamide adenine dinucleotide

 NAD+ and NADP+ ⇒ cofactors in oxidation-reduction
reactions→→ e.g. glycolysis, oxidative
phosphorylation, etc.
Niacin B3
 Deficiency: pellagra
Niacin B3 quantitation

 Normal adults excrete 20 – 30% of their niacin in the

methylnicotinamide form and 40 – 60% in the pyridone
 If ratio of pyridone to methylnicotinamide < 1.0 ⇒ not
 Pyridone is absent for weeks before clinical signs are
 Methylnicotinamide falls to a minimum at about the
time the signs are evident
Niacin B3 quantitation

 Measure metabolites of niacin excretion:

 N(1)-methylnicotinamide

 N(1)-methyl-3-carboxamide-6-pyridone

 This determination done by HPLC


 Assay semi quantitatively with sulfanilic acid →

yields a yellow colour
 Intensity of the yellow correlates with the amount of
niacin present, which is measured against a set of
 Or – colorimetrically use cyanogen bromide as the
colour reagent

 Most sensitive method

 Lactobacillus plantarum
 Growth of organism correlates with the amount of
vitamin in the sample
 The growth response measured by measuring turbidity
Vitamin B6

 3 forms
 Pyridoxine (pyridoxol)

 Pyridoxal – aldehyde

 Pyridoxamine – amine

 Widely distributed in food; dietary deficiency rare

 Decomposed by UV light
Vitamin B6
 Pyridoxal phosphate – formed in liver → cofactor for
transaminases and decarboxylation of amino acids
 Pyridoxine required for:
 Balancing of hormonal changes in women

 Assisting the immune system

 Growth of new cells

 Processing and metabolism of proteins, fats and

 Balancing of sodium and potassium

 Etc.
Vitamin B6

 In women ⇒ pre-menstrual fluid retention, severe

period pains, emotional PMS symptoms,
premenstrual acne, nausea in early pregnancy
 Deficiency: may cause roughening of skin,
peripheral neuropathy, sore tongue
Vitamin B6 quantitation

 Urine determination from 24 hour samples

 Plasma levels are measured by
 Flourometric assays

 After condensation with fluorophore


 Pyridoxic acid (level drops during deficiency)

 Microbiological assay using Saccharomyces uvarum

which measures free B6
Folate / Folic acid

 Present in green vegetables and some meats

 Easily destroyed during cooking →→ dietary
 Absorption through small intestine
 Active form = tetrahydrofolate ⇒ important in purine
and pyrimidine synthesis
 Note: methotrexate (cytotoxic folate analogue)
competes with it for metabolism, therefore inhibiting
DNA synthesis
Folic acid

 Deficiency:
 Common in intestinal malabsorption syndromes,
e.g. ‘contaminated bowel’ syndrome
 During pregnancy and lactation

 Low red-cell folate concentrations ⇒ associated with

megaloblastic anaemia
Folic acid quantitation

 Microbiological assay
 Streptococcus faetalis, Lactobacillus casei
 Liberate free biotin by proteolytic digestion

 Add aliquot to biotin deficient medium inoculated

with test organism
 Derive calibration curve from growth with
calibrators containing known amounts of biotin
Folic acid quantitation

 Other methods:
 Competitive protein binding

 Folate competes with I125 labelled folate for

binding sites on b-lactoglobulin binder and B12
competes with Co-labeled cobalamin for binding
sites on the intrinsic factor
 Immunometric method
Vitamin B12
 Cobalamins
 Found in animal products but not green vegetables
 Dietary deficiency rare
 Absorbed in terminal ileum, combined with intrinsic
factor derived from gastric parietal cells
 Cobalamin transport in plasma → specific carrier
protein: transcobalamin II
 Coenzyme activity in nucleic acid synthesis
Vitamin B12

 Note: Vitamin B12 can only be absorbed when it has

formed a complex with the intrinsic factor
 Complex is resistant to proteolytic digestion
 There is competition with intestinal bacteria, which
also need vitamin B12 for growth
 Adequate absorption depends on normal:
 Gastric secretion of intrinsic factor
 Intestinal mucosa in the distal ileum
 Intestinal bacterial flora
Vitamin B12

 Deficiency → megaloblastic anaemia

 Can cause subacute combined degeneration of the
spinal cord
 Anaemia reversible by treating with folate
 Except – in pernicious anaemia, when such
treatment might even aggravate neurological
lesions, leading to permanent disability
Vitamin B12 quantitation

 Microbiologically:
 Lactobacillus leichmannii
Pantothenic acid

 Pantothenate ⇒ component of coenzyme A (CoA)

 CoA – essential for fat and carbohydrate
 Essential in synthesis of hormones and cholesterol
 Vitamin is widely distributed in foodstuff
 Deficiency may not produce clear syndrome
Pantothenic acid quantitation
 Urinary output
 Urinary output is directly proportional to dietary
 Urinary output of < 1mg/day is considered
abnormally low
 Microbiological assays using Saccharomyces
carlbergensis and Lactobacillus plantarum
 GC
 Enzymatic assay to determine CoA and ACP
Vitamin C

 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

 Normal collagen formation

 Deficiency → scurvy: haemorrhaging at the gums,

large ecchymoses (bruising under the skin)
Vitamin C

 Clinical effects of deficiency:

 Fragile vascular walls → bleeding tendency ↑↑
 Poor wound healing
 Deficiency of bone matrix →→ osteoporosis, poor
healing of fractures
 Anaemia due to impaired erythropoiesis

 Diagnosis of deficiency must be made before any

therapy is begun
 Once therapeutic ascorbate given, it is difficult to
prove any deficiency was previously present
Vitamin C quantitation
 Most methods rely on the reductive properties of
ascorbic acid
 Reduction of:
 2,4-dinitrophenylhydrazine to hydrazone
 2,4- dichlorophenol-indophenol to its colourless
 The principle of this method is titration with
dichlorophenolindophenol (or phenol-indo-2:6-
dichlorophenol, also known as DCPIP)
Vitamin C quantitation

 Ascorbic acid reacts with DCPIP – changing the

colour from blue → colourless
 Reaction is of a 1:1 fashion, so if a known quantity
of DCPIP solution reacts with the plant tissue
extract, the quantity of DCPIP used gives a direct
measure of the quantity of ascorbic acid present
 Not suitable for highly colored products

 2-4 dinitrophenylhydradrazineto forms red bis-

hydrazone or
 2,4-dichlorophenol-indolphenol which is reduced
to a colourless form

 Oxidation of ascorbic acid to dehydroascorbic acid

which reacts with phenylene diamineto to produce a
fluorescent compound
 Intensity is proportional to the vitamin concentration

 Plasma( oxalate, heparin or EDTA)

 Deproteinized sample is stable for 2 months at

 Whole blood
 Leukocytes
 Leukocyte assay immediately

 Urine
Vitamin D

 Fat-soluble
 Manufactured by body on exposure to sun
 Derived from:
 Cholecalciferol (vitamin D )

 Ergocalciferol (vitamin D2)

 Hydroxylation in the liver:
cholecalciferol 25-hydroxycholecalciferol
( 25-OHD3)
Vitamin D

 25-OHD3 is the main circulating form and store of

Vitamin D
 Second hydroxylation in proximal renal tubular cells
of the kidney:
25-OHD3 1,25-dihydroxycholecalciferol
( 1,25-(OH)2D3)
Active metabolite
Vitamin D

 1α-hydroxylase activity and 1,25-(OH)2D3 production

may be stimulated by:
 Low plasma phosphate concentration

 Increase in plasma PTH concentration, possibly

due to lower phosphate
 Estrogens, prolactin, growth hormone ⇒ to
increase calcium absorption during pregnancy,
lactation, growth
Vitamin D
 1α-hydroxylase activity and 1,25-(OH)2D3 production
may be inhibited by:
 Hyperphosphataemia

 Free-ionised hypercalcaemia

 Very close relationship between PTH and 1,25-

(OH)2D3 – both hormones have synergistic action on
osteoclasts ⇒ calcium release into circulation
 In the absence of 1,25-(OH)2D3, action of PTH on
bone is impaired
Vitamin D

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
Vitamin D quantitation

 Cholecalciferol and ergocalciferol

 White, odourless crystals that are soluble in fats and
organic solvents
 The absorption maximum of both in hexane is at
264.5 nm
Vitamin D quantitation
 Only 25-(OH)D3 and 1,25-(OH)2D3 measurement
are of clinical value
 Vitamin D
 Has short half life (1 – 2 days)

 Fluctuates with exposure to sunlight and dietary

 25-(OH)D3
 Used to determine vitamin D status
 Is present in much greater concentrations

 Easier to assay
Vitamin D quantitation

 Measurement of 1,25-dihydroxy vitamin D (1,25-

(OH)2D3 ) is useful for:
 Detecting states of inadequate or excessive
hormone production in the evaluation of
 Hypercalcemia
 Hypercalciuria
 Hypocalcemia
 Bone and mineral disorders
Vitamin D quantitation

 Whether measuring 25-(OH)D3 or 1,25-

(OH)2D3 ,
D2 and D3 should be measured together
 No point in measuring them separately
Vitamin D quantitation
 3 steps
 Extraction
 Frees and partially purifies the metabolite that is
almost completely associated with D transport
protein (DBP) and albumin
 Purification
 Separates vitamin D metabolites, lipids and
interfering substances
 Quantitation
 By competitive binding (RIA)
 UV absorption
 Methods that detect multiple metabolites should not be
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

 require skill and HPLC/UV detector setup

 large sample

 Competitive binding assay

Vitamin D quantitation
 Competitive binding assay
 Vitamin D transport protein (DBP) in serum used as
the specific binder – rat serum provides the DBP
 Titrated 25(OH)D is used as tracer

 Popular method
 Antiserum reacts on equimolar basis with both D2
and D3

 HPLC and UV absorption

 Time consuming and tedious
 Pre-HPLC chromatography step necessary
Vitamin D quantitation
 Determination of 1,25(OH)2D3
 Radioreceptorx Assay
 Acetonitrile deproteinization

 Pass through a commercial column

chromatography cartridge
 Competitive binding assay with Vitamin D
 Not as successful
Vitamin E

 α-tocopherol
 Soluble in fat solvents but not in water
 Sufficient intake with a healthy diet
 Deficiency rare because of easy availability in
 Excess can be harmful
 Known as a potent antioxidant
Vitamin E

 Newborn infants are deficient in vitamin E –

which may be associated with haemolytic
 Especially in premature infants
 Deficiency in adults may be due to
prolonged and severe fat malabsorption
→→ neurological symptoms
Vitamin E

 Viscous oil at room temperature

 Can be measured spectrophotometrically
 However, HPLC method with fluorescence
detection is preferred as it permits the
measurement of different forms of vitamin E
Vitamin E quantitation

 Solvent extraction
 Chromatography
 2D reverse-phase paper, thin layer, column
 Gas chromatography,

 HPLC provides separation of various

Vitamin E quantitation

 Chemical methods
 Based on redox reaction

 Oxidize tocopherol with FeCl to produce red

 Monitor vitamin E status by degree of erythrocyte
 After treatment of cell with hydrogen peroxide (or
 When possible do both
Vitamin K
 Fat soluble
 Also known as the “clotting vitamin” ⇒ without
vitamin K, blood would not clot
 Available in diet as green leafy vegetables, beans,
 Also synthesized by bacteria that line the
gastrointestinal tract, in the ileum
 Deficiency rare
 Deficient →→ increased propensity to bleeding and
Coagulation Cascade
Vitamin K
• Vitamin K is a cofactor
needed for synthesis (in
the liver) of:
• Prothrombin (Factor II)
• Factor 7 (VII)
• Factor 9 (IX)
• Factor 10 (X)
• Proteins C and S

∀ ∴ deficiency of Vitamin K
⇒ predispose to bleeding
Vitamin K

• A deficiency of Vitamin K predisposes to bleeding

• Conversely, blocking the action of vitamin K helps
to prevent inappropriate clotting
• Warfarin is sometimes prescribed as a "blood
thinner" because it is an effective vitamin K
• (Warfarin is also used as a rat poison because it
can cause lethal (internal) bleeding when eaten)
Vitamin K quantitation

 Phylloquinones and menaquinones

 Dissolves in organic fat
 Destroyed by alkaline solution and reducing agents
and sensitive to UV light
 Determination requires 3 steps
 Extraction

 Chromatographic separation

 Quantitation
Vitamin K quantitation

 Photometric determination after reaction with

one of reactive compounds like
phenylhydrazine, piperidine
 Spectrophotometric determination of
chemically reduced vitamin K
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

 Dietary deficiency does not occur – synthesis by

bacteria in ileum
 Deficiency:
 In patients with steatorrhoea → vitamin cannot be
absorbed normally
 Following administration of broad-spectrum
antibiotics → alter intestinal bacterial flora,
reducing vitamin K synthesis
Vitamin K deficiency

 Newborn plasma [vitamin K] lower than in adults

 Very little transported across the placenta

 Neonatal gut only gradually colonised by vitamin

K synthesizing bacteria
 Protein synthesis has not reached full adult
 Severe deficiency → haemorrhagic disease of the
Vitamin quantitation

 Physical activities, stress, temperature, and some

therapeutic drugs can make borderline interpretation
 Impaired fat absorption
 Fat excretion > 5 g/day
 Foul-smelling, bulky, pale, greasy stools
 Intestinal malabsorption: fat digestion normal but
impaired absorption of products of digestion
 Pancreatic steatorrhoea – absorptive capacity
normal but fat cannot be digested because of
deficiency of digestive enzymes

The container is filled with yellow, liquid stool

that contains a high level of fat

Dr Ralph Leischner