Comprehensive Biochemistry for Dentistry Textbook for Dental Students

276 10 Hormones

Metabolic Functions
Preparation of Uterus for Implantation

• Progesterone increases synthesis of glycogen in endometrial cells. It increases lipid
store of endometrium. It boosts protein synthesis and activates proliferation of
endometrial cells. It increases proliferation of glandular tissues in endometrium.

• Overall, progesterone prepares uterus for implantation after fertilization.
• Progesterone is a pregnancy hormone.
• Progesterone inhibits secretion of luteinizing hormone (LH) from anterior

pituitary. It suppresses ovulation, and
• progesterone helps in the development of the breast and mammary glands.

It promotes development and growth of alveolar ducts in mammary glands. It
prepares mammary glands for lactation after child birth.
• Progesterone has thermogenic effect. It increases body temperature during
ovulation.

Placental Hormones
The placenta is a connecting organ between a growing fetus and the wall of uterus
in a mother’s body. It ensures gaseous exchange, blood supply, and waste elimina-
tion. Placenta also serves as endocrine organ.

Placenta secretes two hormones as follows:
Human chorionic gonadotropin hormone (HCG)

Chemistry
HCG is a glycoprotein.

Protein part of hormone is a heterodimer. It is made up of two subunits as:

• Alpha-chain
It is composed of 92 amino acid residues. This subunit is identical to LH, TSH,
and FSH hormones from the pituitary gland.

• Beta-chain
It is made up of 145 amino acid residues.

Carbohydrate part of hormone is composed of oligosaccharides which are
attached to asparagine and serine residues.

Biosynthesis
It is synthesized by syncytiotrophoblasts in the placenta. Syncytiotrophoblasts con-
stitute the outermost layer of cells which invade the wall of the uterus.

Metabolic Functions

• Maintenance of Corpus Luteum
HCG hormone acts on LHCG receptors in the ovary. It induces maintenance of
corpus luteum in the first trimester of pregnancy. It regulates secretion of proges-
terone through corpus luteum.

10.16  Applied Biochemistry 277

• Secretion of Testosterone
HCG stimulates growth of Leydig cells in fetal embryo. It induces secretion of
testosterone from fetal testes.

Normal Serum Value
Free testosterone

• Its value in males is 8–30 ng/100 mL and in females is up to 2 ng/100 mL.

Total Testosterone

• Its value in males is 280–1000 ng/100 mL and in females is 20–75 ng/100 mL.

Progesterone

• Its value in males is <1 ng/mL and in females during pregnancy is between
10 and 80 ng/mL.

10.16 A pplied Biochemistry

Role of Estrogen and Progesterone in Inflammatory Gingivitis
During pregnancy, serum levels of estrogen and progesterone are elevated. These
hormones have inflammatory effect on the gingiva and periodontia.

• Elevated levels of progesterone and estrogen promote growth of microflora in
oral cavity. These hormones induce pro-inflammatory cytokines.

• ↑Progesterone probably alters proliferation of fibroblasts and collagen synthesis.
These changes aggravate inflammation in gingival and compromise repair in gin-
gival tissues in pregnancy.

• Additionally, iron deficiency coupled with folate deficiency in pregnancy further
retard healing of gingival tissues.

• Pregnancy-induced estrogen and progesterone level can cause gingivitis in 60%
of pregnant women. Poor oral hygiene is a chief predisposing factor to gingivitis
and periodontitis in association with increase in progesterone and estrogen levels
in pregnancy in women.

• Furthermore, use of oral contraceptives is also associated with prevalence of
chronic gingivitis and periodontitis in women.

• It is advisable to keep good oral hygiene by proper tooth brushing and use of
antiplaque mouth wash. It helps to minimize proliferation of causative organisms
and control aggravation of already-existing hormonal-induced inflammation in
gingivae and periodontia.

278 10 Hormones

Suggested Readings

Alberti KGMN (ed) (1978) Recent advances in clinical biochemistry. Churchill Livingstone,
London

Astwood EB (1968) Recent progress in hormones research. Academic, New York
Baron DN (1982) A short textbook of chemical pathology, 4th edn. Wiley, New York
Conn EE, Stump PK (1969) Outline of biochemistry, 2nd edn. Wiley, New Delhi
Korenberg A (1980) DNA replication. W. H. Freeman, New York
Zachariasen RD (1993) The effect of elevated ovarian hormones on periodontal health: oral con-

traceptives and pregnancy. Women Health 20(2):21–30

Vitamins 11

11.1 D efinition

Vitamins may be defined as organic compounds that are essential in minute
amounts for the attainment of normal growth and maintenance of general health.

11.2 Important Facts About Vitamins

• In 1912, a biochemist named Casimir Funk extracted a water soluble compound
from rice bran and named it “vitamine.” He thought that it was an “amine” which
was “vital” for life.

• Later research showed that all compounds were not amines. It was proposed by
Jack Cecil Drummond that the suffix “e” should be dropped from vitamine.

• Vitamins are essential nutrients.
• Vitamins differ from macronutrients. They do not produce energy in the body.

They are required in minute amounts unlike macronutrients such as proteins,
lipids, and carbohydrates which are necessary in large amounts.
• Vitamins are micronutrients because they are supplemented in minute amounts.
They are essential for normal physiological functions. Micronutrients include
vitamins and minerals.
• Vitamins differ from hormones. Most of vitamins are supplemented with diet.
Hormones are synthesized in the body by endocrine glands.
• Vitamins differ from enzymes. Enzymes are protein in nature. Enzyme catalyzes
biochemical reactions. Vitamins may act as cofactor in enzymatic reactions.

11.3 C lassification of Vitamins

Based on the solubility, vitamins are classified into two groups as follows:

© Springer Nature Singapore Pte Ltd. 2019 279
A. Gupta, Comprehensive Biochemistry for Dentistry,
https://doi.org/10.1007/978-981-13-1035-5_11

280 11 Vitamins

1. Fat soluble vitamins
• They are soluble in nonpolar organic solvents.
• Bile salts help in the absorption of fat soluble vitamins from the intestine.
• They are stored in the liver.
Types: Vitamins A, D, E, and K

2. Water soluble vitamins
• They are soluble in water.
• They are absorbed easily.
• They are excreted in urine.
Types: Vitamin C and vitamin B complex

11.4 Fat Soluble Vitamins

11.4.1 Vitamin A

Vitamin A includes unsaturated organic compounds essential for living organisms.

History
• In 1816, physiologist F. Magendie observed that poorly nourished dogs devel-

oped corneal ulcer.
• In 1880, N. Lunin demonstrated the presence of a compound in milk that was

vital for nutrition.
• In 1911, W. Stepp proved fat soluble nature of vital substance in milk.
• In 1911, Hopkins proved the presence of vital factor in trace amount in milk that

was essential for nutrition and health. In 1918, vital factor in milk was declared
as vitamin A.
• In 1932, Paul Karrer demonstrated chemical structure of vitamin A.
• 1937, H. Holmes and R. Corbet extracted and crystallized vitamin A.

Chemical Structure
1. Forms of Vitamin A

It exists in nature in three distinct forms:
• Retinol

–– It is called as vitamin A (alcohol). Retinol exists in two forms such as
vitamin A1 and vitamin A2.

–– Vitamin A1 is mainly present in animal sources. It has only one double
bond in β-ionone ring. It has higher biological activity (60%) than vitamin A2.

–– Vitamin A2 (3-dehydro-retinol) is present in the liver of fresh water
fish. It is has two double bonds in β-ionone ring. Its biological activity is
less than half (40%) of the biological activity of vitamin A1.

–– Neo-vitamin A is the stereoisomer of vitamin A1. Neo-vitamin A has 70%
of the biological activity of vitamin A1.

–– Retinol form of vitamin A is predominant in animal body tissues. It is
esterified with long-chain fatty acids into “retinyl ester.”

• Retinal
–– It is called vitamin A aldehyde. Retinal and retinol can be interconverted
by retinal reductase enzyme.

11.4  Fat Soluble Vitamins 281

• Retinoic acid

–– It is called vitamin A acid. It is formed by oxidation of retinal. Retinoic

acid is nonconvertible into retinal or retinol as in Fig. 11.1
All three forms of vitamin A are called “retinoids.” They are found exclu-
sively in animal tissues.
2 . Structure
• Vitamin A has β-Ionone ring (cyclohexenyl) in its structure. It is
2,6,6-t­rimethyl cyclohexenyl ring. The ring contains three methyl groups and

one double bond.
• Isoprenoid chain is attached to β-Ionone ring. The isoprenoid chain has four

double bonds, two methyl groups, and “R” group which is linked to terminal

carbon.
• “R” group can be alcohol, aldehyde, or an organic acid as in Fig. 11.1.
3. Carotenoids (Provitamin A)
• Vitamin A is derived from carotenoids. They are provitamin A.
• Carotenoids are called “tetraterpenoids.” They possess four “terpene” units

(C10) or eight isoprene units and are C40 compounds as in Fig. 11.1.
Carotenoids are pigmented unsaturated organic compounds synthesized by

plants, algae, bacteria, and fungi.
• These compounds have red, yellow, and orange colors. Carotenoids are

exclusively found in plant tissues.
• Carotenoids are classified into two categories of organic compounds. The

compounds which do not contain oxygen are called “carotene.” The other
category of compounds containing oxygen is called “xanthophyll.”
• Carotene is derived from the Latin word carota that means carrot. They

are pigmented unsaturated group of hydrocarbons. Their general molecular
formula is C40Hx. Carotenes are tetraterpenes. Carotene exists in alpha,
beta, and gamma forms.
• Beta-carotene is a deeply reddish orange pigmented compound found in

plants and fruits.

–– It has two beta-ionone rings at both ends of molecule.
–– B eta-carotene is the most prominent carotenoid with “provitamin A”

activity.

–– B eta-carotene molecule is cleavage by beta-carotene 15,15′ oxygenase
(dioxygenase) enzyme into two molecules of vitamin A.

• Alpha-carotene and gamma-carotene.

–– They possess single beta-ionone ring. They have mild (30–40%) provita-

min A activity. They yield only single molecule of vitamin A by beta-car-

otene 15,15′ oxygenase enzyme.
• Beta-cryptoxanthin is another source for provitamin A.

–– It has around 30–40% of provitamin A activity. It yield single molecule of
vitamin A. It is related to xanthophyll category of carotenoids. It is

found in fruits like oranges and tangerines and in human blood.
Beta-ionone ring is absent in other carotenoids. They do not have vitamin
A activity. Animals including humans have capability to convert beta-
ionone ring (retinyl group) of carotene into retinol.

282 11 Vitamins

Site of Oxidation

CH3 CH3 CH3 CH3 CH3 CH3 CH3

CH = CH – C = CH – CH = CH – C = CH – CH =CH – CH = C – CH = CH – CH = C – CH = CH

CH3 CH3 B- Carotene CH3

Oxidation B-Ionone
Ring

B – Carotene dioxygenase

CH3 CH3 CH3

CH = CH – C = CH – CH = CH – C = CH – CHO

CH3 CH3

All trans – retinal

Retinal isomerase

CH3 CH3 11

CH = CH – C = CH – CH

CH3 12 CH

CH3

H3C – C

CH

CH3 CH3 CH3 CHO
11 – CIS – Retinal

CH3
CHO

CH3
RETINAL

NADPH2 REDUCTASE
NADP+

RETINAL

CH3 CH3 CH3 H2O

CH = CH – C = CH – CH = CH – C = CH – CH2OH NAD+

CH3 CH3 NADH2

Retinol

oxidation

RETINAL DEHYDROGENASE
RETINOIC ACID

Fig. 11.1  Interconversion of forms of Vitamin A

11.4  Fat Soluble Vitamins 283
Table 11.1  RDA of vitamin A
Age group RDA for vitamin A (IU retinol)

Children 1500–2000 IU
2500–3000 IU
Adults 3000 IU

Pregnant
women

Lactation 4000 IU

Source: (NIH 2017)
Retinol (1 IU) = 0.3 mcg retinol activity equivalents
Beta-carotene (1  IU)  =  0.6  mcg retinol activity
equivalents

Dietary Sources of Vitamin A
1. Animal Sources

Animal sources of food contain “retinyl palmitate” as a form of vitamin A
Example: milk, cheese, egg yolk, butter, and cod-liver oil.
2 . Plant Sources
Plant sources of food contain carotenoids, chiefly as beta-carotene and beta-­
cryptoxanthin as provitamin A Example: carrots; green leafy vegetables like
spinach, amaranth, broccoli, dandelions green, mustard greens, and lettuce; and
fruits like papaya, mango, oranges, tangerines, and sweet potato.
Spirulina is Cyanobacteria (blue-green algae) and is a seafood. It is a rich
source of vitamin A.

R ecommended Dietary Allowance of Vitamin A
Recommended Dietary Allowance (RDA) is “the average quantity of a nutrient
which is sufficient to fulfill physiological needs of almost all (97.5%) healthy
individuals.”

RDA was developed by a committee comprising Lydia J.  Roberts, Hazel
Stiebeling, and Helen S. Mitchell during the Second World War, and the committee
was set up by the US National Academy of Sciences (Table 11.1).

Absorption, Transport, and Storage of Vitamin A
Site and Process of Absorption

• Intestinal mucosa is the main site for absorption of vitamin A.
• Food contains carotenoids and retinyl ester. Carotenoids are incorporated into

mixed micelles. Carotenoids are absorbed by passive diffusion.
• Retinyl ester is hydrolyzed into retinal by cholesterol ester hydrolase enzyme. It

occurs in lumen of small intestine. Retinal is reduced into retinol by retinal
reductase enzyme. It is incorporated into mixed micelles. Retinol absorption
occurs by active transport (carrier-mediated transport).
• Inside enterocytes, retinol is re-esterified with long-chain fatty acids. Carotenoids
(beta-carotene, alpha-carotene, and beta-cryptoxanthin) are metabolized by beta-
carotene dioxygenase enzyme into retinol. The re-esterified retinol is released
into lacteals as chylomicrons. Retinyl ester is transported to the liver as in
Fig. 11.2.

284 11 Vitamins

VITAMIN A

Food Brush border surface
β – Carotene
Enterocyte

ROD Cell Retinylesters β – Carotene
FFA
All-Trans-Retinol O2
All-Trans-Retinal FFA Retino [Free Fatty Acid] Retinal

II-Cis-Retinal Distribured Reduction
Retinol
Retinyl ester

Chylomicrons

Lacteals
circulation

Nucleus Blood
circulation

Retinol All-Tras-Retinol Liver
+
Retinol binding Retinyl
(RBP) protein palmitate

Storage form of Vit. A

Retinol-RBP
complex
RBP

Retinol Target cell

Retinic acid

Nuclear Retinol Nucleus
receptor
Transcription

Regulate cell function

Fig. 11.2  Vitamin A absorption, transport and regulatory function

Storage

• Vitamin A is stored in the liver as “retinyl palmitate.” Around 95% of vitamin A
is stored in the liver.

• It is released as retinol in blood circulation. Its normal plasma level is 18–60 μg
per 100 ml of blood.

11.4  Fat Soluble Vitamins 285

Transport of Vitamin A in the Blood

• Retinol is transported in the blood in bound state. It binds with “retinol binding
protein” (RBP) in plasma and forms complex.

• Retinol-RBP complex circulates in the blood. It is carried to target cells. This
complex is attached to specific receptors on target cells. Retinol enters the
cell.

• Inside the cytoplasm, retinol binds to “retinoic acid binding protein,” and the
complex is carried to nuclear receptor on the surface of nuclear membrane. It
activates genes and controls cellular metabolism.

• Retinol acts similar to steroid hormone.

F unctions of Vitamin A
Vitamin A has multiple functions. Its different forms have diverse functions. Retinal
is necessary for normal vision. Retinol acts as a steroidal hormone, and it regulates
protein synthesis. Retinol is necessary for cell differentiation and normal growth.
Retinoic acid is helpful in glycoprotein synthesis.

Diverse functions of vitamin A are described as follows:

Role of Vitamin A in Vision
Vitamin A contributes to structural integrity of photosensitive pigments of rods and

cones. It plays an important role in the “visual cycle” or “visual

phototransduction.”
Visual cycle or “visual phototransduction” is an elaborate neurochemical,

enzyme-controlled, cyclic process in which light energy is converted into elec-
trical energy (nerve impulse) by photoreceptors.

It was “George Wald” who described visual cycle in 1968. The visual cycle is
called “Wald’s visual cycle” or “rhodopsin cycle.”

Types of Photoreceptors in Human Eye
• The retina of the human eye contains two types of photoreceptors such as rods

and cones.
• Rods contain photosensitive pigment called “rhodopsin.” It is also called “visual

purple.” This word is derived from Greek words “rhodon” for rose due to pink

color and “opsis” for sight. Rods appear red due to rhodopsin.

• Rods have very high sensitivity to light, and they are helpful in vision in dim
light. This type of vision is called “night vision” or “scotopic vision.”

• Cones contain photosensitive pigment called “conopsin.”
–– Depending on type of conopsin, cones have three types.
–– Cones with “porphyropsin” are sensitive to red color at a wavelength of

665 nm.
–– Cones with “iodopsin” are sensitive to green light at a wavelength of 535 nm.
–– Cones with “cyanopsin” are sensitive to blue light at a wavelength of 445 nm.

–– Conopsin is sensitive to bright light, and it is helpful in vision in bright light.

This type of vision is called “color vision” or “photopic vision.”

286 11 Vitamins

Elements of WALD’s Visual Cycle
Structure of Rhodopsin

• Rhodopsin is a light-sensitive conjugated protein. It is located within membra-
nous discs in rods. Rhodopsin is composed of a protein called “opsin” and a
prosthetic group “retinal.”

• Opsin is a G-protein-coupled receptor. It is embedded in phospholipid layer of
membranous discs. It has seven transmembrane helices. Opsin is not
photosensitive.

• Retinal is a photosensitive moiety in rhodopsin molecule. It is also called “reti-
nene1.” The rhodopsin contains 11-cis-retinal as an isomer of retinal. It is
enclosed within seven transmembrane helices of opsin. The 11-cis-retinal is
linked covalently to amino group (–NH2) group of lysine residue in opsin with
protonated Schiff’s base (–NH+〓CH–).

• Dietary vitamin A and carotenoids are necessary for biosynthesis of
11-cis-retinal.

Photochemical Transformation in Rhodopsin

• A photon of light falls on rhodopsin. Light energy is absorbed by 11-cis-retinal,
and it undergoes photoisomerization into all-trans-retinal.

• The all-trans-retinal induces a series of conformational changes in opsin mole-
cule. A number of intermediates are produced. The unstable intermediates are as
follows:
–– Photorhodopsin is produced within femtoseconds (10−15 s).
–– Bathorhodopsin is produced within picoseconds (10−12 s).
–– Lumirhodopsin.
–– Metarhodopsin I.
–– Metarhodopsin II (photoactivated rhodopsin) contains deprotonated
Schiff’s base which is linked to all-trans-retinal. Its color changes from red
to yellow. Metarhodopsin II initiates phototransduction.

• The metarhodopsin II activates G-protein located inside the cytosol. It splits into
scotopsin and all-trans-retinal as in Fig. 11.3.

Phototransduction

• The G- proteins are guanine nucleotide-bound proteins. They are considered
as “molecular switches” present inside the cytosol. They help to transmit signals
from a stimulus outside the cell membrane to inside the cytosol.

• In rods and cones, “transducin” is the G-protein. It is composed of α-subunit,
β-subunit, and γ-subunit. These subunits are linked to internal surface of cell
membrane of rods.

• Activated transducin is bound to GTP, and it undergoes hydrolysis into GDP
with release of inorganic phosphate as in Fig. 11.3.

11.4  Fat Soluble Vitamins 287
Light
VITAMIN A

Rhodopsin Batho Rhodopsin
Opsin + II CIS RETINAL Lumi Rhodopsin

In Meta Rhodopsin I
Darkness
Meta Rhodopsin II
with in
Retina

OPSIN Within
Retina

Within retina Retinal Within All Trans Retinal
II – CIS – Retinal Isomerase Retina in
Darkness Enters
Blood circulation
NADH2
Reaches Retina
Acohol dehydrogenaseNAD+Reaches
Liver

WALD’S Within liver
VISUAL CYCLE
All – Trans Retinal
Enters blood circulation Liver Retinal isomerase
Fig. 11.3  Wald’s visual cycle
II – CIS Retinol

Liver NAD+ Acohol
dehydrogenase

NADH2

II – CIS Retinol

288 11 Vitamins

• Transducin enters the cytosol and activates cyclic GMP phosphodiesterase
(cGMP) enzyme.

• Activated cGMP phosphodiesterase enzyme cleavages phosphodiester bonds in
cGMP. The enzyme hydrolyzes cGMP into 5’ GMP.

• The cGMP is the second messenger of cell. It is present in the cytosol of cells.
It helps to regulate physiological activities of cell.

• Cell membranes of rods have sodium ion channels. They are cGMP-gated.
• In darkness, high concentration of cGMP in rods always keeps the sodium

channels in open state. The sodium ions enter inside the rods through sodium
channels.
• “Dark current.” It is the constant influx of sodium ions inside rods. It is because
the sodium influx is maintained only when the rods are not stimulated as in dark-
ness. Sodium influx is responsible for a resting membrane potential of −40 mV
in rods. This is a unique characteristic of rods. Otherwise, resting membrane
potential of other sensory cells in the body is between −70  mV and
−90 mV. Therefore, sodium influx always maintains a state of mild depolariza-
tion in rods.
• Depolarized rods release glutamate neurotransmitter. The bipolar and ganglionic
cells in retina do not generate action potential.
• In light, activated cGMP phosphodiesterase enzyme cleavages phosphodiester
bonds in cGMP. The enzyme hydrolyzes cGMP into 5′ GMP, and concentration
of cGMP in cytosol of rods is decreased.
• A decline in cGMP concentration in cytosol closes the sodium channels. The
influx of sodium ions is stopped. As a result, resting membrane potential in rods
increases to −70 mV. This rise in electrical potential in cell membrane of rods is
called “hyperpolarization.”

Hyperpolarization in Rods

• Hyperpolarization results in closure of voltage-gated calcium channels in rods.
The release of glutamate at rods-bipolar cell synapse is inhibited.

• Inhibition of glutamate depolarizes bipolar cells in the retina. They release neu-
rotransmitter at bipolar-ganglionic cell synapse. This leads to generation of
action potential in ganglionic cells.

• The impulse (action potential) from ganglionic cells reaches optic nerve. It is
transmitted to visual center in cerebral cortex by optic nerve as in Fig. 11.3.

Regeneration of Rhodopsin
Rhodopsin is regenerated in the retina and liver through the action of enzymes.

• In the retina
–– This process occurs in darkness. The all-trans-retinol undergoes isomeriza-
tion into 11-cis-retinal by the activity of retinal isomerase enzyme.
–– 11-cis-retinal recombines with opsin to form rhodopsin.

11.4  Fat Soluble Vitamins 289

• In the liver
–– The all-trans-retinal enters into blood circulation and is transported to the
liver.
–– It is reduced to all-trans-retinol by alcohol dehydrogenase enzyme in the
liver. The reduction requires NAD+ coenzyme and zinc as cofactor.
–– The all-trans-retinol is isomerized into 11-cis-retinol by retinol isomerase
enzyme.
–– 11-Cis-retinol is oxidized into 11-cis-retinal by dehydrogenase enzyme in the
presence of coenzyme, NADH2.
–– 11-Cis-retinal enters blood circulation and transported to the retina, and it
recombines with opsin to form rhodopsin.

Regeneration of rhodopsin completes the Wald’s visual cycle. Similar events
occur during phototransduction in cones.

Role in Color Vision
• Cones possess conopsin. It is composed of opsin and retinal moiety. Therefore,

vitamin A is necessary for daylight vision and color vision.

Role in Normal Epithelialization
• Retinol is necessary for normal epithelialization in the skin and mucous mem-

brane. It prevents excessive keratinization.

Role in Mucus Formation
• Retinyl phosphate is helpful in the formation of glycoproteins. These are struc-

tural components of mucus which is secreted by mucous membranes in body
cavities. Mucus keeps the surface of mucosa moist and healthy. Vitamin A pro-
tects mucosa from infection.

Role in Growth
• Retinoic acid behaves like steroid hormone. It binds to nuclear receptors. The

all-trans-retinoic acid binds to “retinoic acid receptors” (RARs), and 9-cis-­
retinoic acid binds to “retinoid X receptor.” Retinoic acid regulates transcription

of genes. So retinoic acid regulates protein synthesis in the body.

• Therefore, retinoic acid is necessary for cell differentiation and growth of cells in

the body.

Role in Transferrin Synthesis
• The all-trans-retinoic acid has found to regulate protein synthesis. It enhances

synthesis of transferrin protein. It binds with iron in blood circulation.

Role in Immunity
• Vitamin A plays an important role in the development of immune system. It is

necessary for appropriate immune response against various infections.
• In experimental model on mice, it has been found that retinoic acid stimulates

proliferation and cytotoxicity of T cells.

290 11 Vitamins

• Retinoic acid can modulate activity of antigen-presenting cell.
• Retinoic acid can stimulate proliferation of B cells and antibody formation.

Role in Normal Reproduction
• The all-trans-retinoic acid is necessary for normal reproduction. The fact has

been proven by experiments on vitamin A-deficient (VAD) male and female rats.
The development of testes in VAD male rats was affected. The VAD female rats
were found to be sterile.
• Vitamin A is also necessary for normal embryo development.

Role in formation of Bones and Teeth
• Vitamin A has a role in the formation of bones and teeth. Retinoic acid activates

osteoblastic activity, and its deficiency decreases endochondral ossification.

Vitamin A as Antioxidant
• Antioxidant is a chemical compound that inhibits oxidation of biomolecules in

living system. They prevent lipid peroxidation and damage to proteins and
DNA.  The beta-carotene scavenges free radicals in the body and acts as
antioxidants.

Vitamin A Has Anticancer Activity
• The all-trans-retinoic acid (ATRA) has found to have anticancer activity. It can

enhance anticancer effect of epigallocatechin-3-O-gallate (phytochemical in
green tea) on subcutaneous growth in mice model.
• The use of all-trans-retinoic acid has been approved by the US Food and Drug
Administration for the treatment of lymphoma and melanoma. The (ATRA) is
found to inhibit growth of tumor and metastasis.

Vitamin A Deficiency Disorders
1. Night blindness.

• Night blindness is the inability of an individual to see in dim light. It is the
initial manifestation of VAD.

• For normal visual cycle, a constant supply of vitamin A is necessary. It helps
to regenerate rhodopsin in the retina. Deficiency of vitamin A impairs rhodop-
sin regeneration and impairs the dark adaptation. It causes night blindness. It
is also called “nyctalopia.”

2. Xerophthalmia.
• It is characterized by dryness of the conjunctiva and cornea. Vitamin A defi-
ciency results in structural changes in lachrymal glands causing
xerophthalmia.

3. Bitot’s spot.
• Bitot’s spots are white, opaque, and triangular- or irregular-shaped lesions in
conjunctiva. They occur on either outer or inner side of the eye. It is due to
localized keratinization of conjunctiva. Lesion is named after French physi-
cian Pierre Bitot who described the lesion in 1863.

11.4  Fat Soluble Vitamins 291

4. Keratomalacia.
• Corneal epithelium becomes keratinized and opaque. Keratomalacia is a
severe form of xerophthalmia which starts as xerosis of conjunctiva. This
condition progresses to xerosis of the cornea.
• Cornea undergoes ulceration and necrosis. The condition is irreversible.
• Keratomalacia is the leading cause of blindness among children in developing
world. VAD-associated blindness is preventable.

5 . Deficiency of vitamin A causes keratinization in mucosa of respiratory tract.
The respiratory tract is susceptible to higher incidence of infections.

6. Deficiency of vitamin A leads to keratinization of urinary tract mucosa. It raises
incidence of calculi formation in the urinary tract.

7 . Sterility in males.
• In mice model, VAD results in degeneration of germinal epithelium. It is
the cause of male sterility.

8 . The skin becomes dry, scaly, and rough.

H ypervitaminosis A
It is the toxicity associated with consumption of excessive amount of vitamin A. It
is associated with following clinical manifestations:

• Hypervitaminosis A induces osteoclastic activity. Bone resorption and bone pain
are common symptoms. In infants and children, thinning of the skull bone (cra-
niotabes) occurs.

• Enlargement of the liver, nausea, and vomiting.
• Intracranial pressure is increased, headache.
• Incidence of fetal malformation in pregnant women.
• Yellow discoloration of the skin, peeling of the skin, hair loss, and pruritus.
• Loss of weight.

A pplied Biochemistry
Role of Vitamin A in Oral Leukoplakia
Oral leukoplakia, according to WHO, can be defined as “a white patch which can-
not be diagnosed clinically or histopathologically as any other oral lesion.”

The cause of oral leukoplakia is uncertain. Smoking is associated with higher
incidence of the lesion. Chronic irritation in the mouth, alcohol consumption,
human papilloma virus, Candida albicans, and vitamin deficiency are the leading
predisposing factors for the disorder.

Oral leukoplakia has a premalignant potential without a particular histological
pattern.

Treatments of oral leukoplakia are dependent on the extension of lesion and its
histological pattern.

Surgical excision is indicated for localized lesions.
Retinoids are new drugs derived from vitamin A. They are administered orally
and applied topically. Retinoids act by inducing apoptosis of cancer cells.

292 11 Vitamins

The isotretinoin, 13-cis-retinoic acid, is vitamin A analog. It has the highest
potential in the prophylaxis of secondary tumors.

According to a study by Epstein and Gorsky (1999), 26 patients were adminis-
tered topically 0.05% tretinoin ointment four times a day, and it was continued for
3.5 years. Around seven to eight patients were cured of oral leukoplakia, and ten
patients experienced recurrence of lesion after the treatment was stopped.

According to a study by Piattelli et al. (1999), ointment containing 0.1% 13-cis-­
retinoic acid was applied topically for 3 months. One patient was relived from the
lesion.

11.4.2  Vitamin D

History
• In 1919, Mellanby found that cod-liver oil has potential to prevent rickets in

experimental models on dogs.
• In 1922, McCollum named active ingredient of cod-liver oil as “vitamin D.”
• In 1931, Augus and coworkers isolated vitamin D and named “calciferol.”
• In 1950, its chemical structure was discovered by Otto Diels and Kurt Alders and

awarded Nobel Prize.

Chemical Structure
1. Forms of Vitamin D

• Vitamin D exists in five different forms such as vitamins D1, D2, D3, D4, and
D5. However, vitamins D2 and D3 are biologically important forms.

• Vitamin D1 is a combination of ergocalciferol and lumisterol in 1:1 ratio.
• Vitamin D2 is “ergocalciferol.”

–– It is synthesized from “ergosterol.”
–– E rgosterol is a sterol found in “ergot” fungi and yeast. The ergot fungi

belong to genus Claviceps. Ergosterol is a normal structural constituent of
cell membrane of fungi and yeast.
–– T he plant “alfalfa” contains ergosterol. It is a perennial plant with flowers.
It is used as forage crop in North America and in countries like New
Zealand and South Africa.
–– E rgosterol is “provitamin D2.” It can absorb UVB (medium wave UV
rays with 290–315 nm wavelength) radiation. It undergoes photolysis.
–– P hotolysis is characterized by activation of double bonds in B-ring due to
absorption of light photon. There is cleavage of bond between C9 and C10
and opening up of B-ring at C9–C10 position. It forms 9,10-secosteroid,
called “previtamin D2.”
–– P revitamin D2 exists in thermodynamically unstable cis and stable trans
forms. The cis form undergoes intramolecular rearrangement and con-
verted into “vitamin D2.”

11.4  Fat Soluble Vitamins 293

• Vitamin D3 is “cholecalciferol.”
–– It is synthesized from “7-dehydrocholesterol.”
–– 7-dehydrocholesterol is the natural zoosterol. It is found in the epider-

mis (stratum basale and stratum spinosum) of the human skin. It is also

present in mammalian milk and insects
–– 7-dehydrocholesterol is provitamin D3. The UVB radiation can pene-

trate into the epidermis only. The 7-dehydrocholesterol can absorb UVB

radiation (290–315 nm wave length) and undergoes photolysis.

–– I t is converted into thermodynamically unstable form, previtamin D3. It is
spontaneously photoisomerized into stable form, vitamin D3.

–– From the epidermis, vitamin D3 passes into capillaries in the dermis, and it
is transported to the liver.

• Active vitamin D3 is “calcitriol” (1,25-dihydroxycholecalciferol).
• Vitamin D4 is “22-dihydroergocalciferol.” It is found in mushrooms.
• Vitamin D5 is “sitocalciferol.” It is an important phytosterol.
“7-Dehydrocholesterol” and “ergocalciferol” are together referred to as
“provitamin D.”
2 . Structure

• Vitamin D belongs to fat soluble group of solid alcohols with fragmented

steroidal ring structure. These are called as “secosteroids.” The word is
derived from Latin words secare meaning to cut and stere for steroid mean-
ing solid and ol meaning alcohol as in Fig. 11.4.

• Secosteroids are subgroup of steroids. These are classified based on the cleav-

age of bond between carbon atoms. Cholecalciferol is 9,10-secosteroids. It

has broken bond between C9 and C10in B-ring.
• Provitamins D2 and D3 have some common structural features:

–– Both provitamins are C28 organic compound with molecular formula as
(C28H43OH).

–– They have “cyclopentano-perhydro-phenanthrene nucleus” and are made

up of four rings named as A, B, C, and D.

–– Hydroxyl group (OH) at C3.
–– C17 has a hydrocarbon chain.
–– Two double bonds between C5–C6 and C7–C8.
–– Ergocalciferol differs from cholecalciferol with the presence of addi-

tional methyl group at C24 and a double bond between C22 and C23 in
hydrocarbon chain as in Fig. 11.4.

D ietary Sources of Vitamin D
1 . Animal Sources

• Richest source: Fish liver oil is the best source of vitamin D3. The oil derived
from the liver of cod fish (cod is the name given to genus the Gadus of fishes)

is the richest source of vitamin D. One spoon full of cod-liver oil contains

around 1400 IU of vitamin D.
• Good source: Flesh of fishes like sardine, salmon, and tuna. Around 90 g of

salmon flesh contains about 450 IU of vitamin D.

294 11 Vitamins

21C 20 C 22 C23 Opening of
Steroid Ring at
12 18 17 C24 C 26 C9-10 Positions
D 16 C 25
11 13 S
C C 27
C 19 Uv Rays
19
2 10 B 8
A
HO 3 4 5
7

6 19
CH2
7 – Dehydrocholesterol
57
[Present in Epidermis] 6

HO

26
18 25

27

in Skin Secosterol
[Cis form]
C5 19
CH3

C 1
HO 3 C2

Cholecalciferol (D3) REenatcehrsesBlLoiovedr, undergoes HEynd2zr5yomxHyeylsadtr–ioolLnaisvoeefsr25C

[Trans form]

25 OH
C5 CH2

31 1 – ∝ Hydroxylase Enters circulations 25 OH
HO in C
2 OH
Kidneys Reaches Kidneys CH2

1;25 – Di hydroxy
cholecalciferol

[Calcitriol]

3 25 – Hydroxy cholecalciferol
HO

Fig. 11.4  Synthesis of Calcitriol (active Vitamin D3)

• Average source: Egg yolk (1 egg) and the liver of beef (80 g) contain around
40  IU of vitamin D.  The unfortified whole milk (250  ml) contains around
40 IU of vitamin D.

• Poor source: Cheese (30 g) contains around 5 IU of vitamin D.

11.4  Fat Soluble Vitamins 295

Table 11.2  RDA of Age group RDA for vitamin D (IU)
vitamin D
Children 400–600 IU
Adult males 600 IU
Adult females 600 IU
Old aged (>50 years) 800 IU

Source: (NIH 2017)
1 IU = 0.025 mcg of cholecalciferol or ergocalciferol

2 . Plant Sources
• Sun-exposed mushroom contains provitamin D2.
• Fortified foods like soya milk, whole cow milk, yogurt, cereals, and orange
juice are good sources of vitamin D for vegans.

R ecommended Dietary Allowance of Vitamin D
Recommended Dietary Allowance (RDA) is “the average quantity of a nutrient which is
sufficient to fulfill the physiological needs of almost all (97.5%) healthy individuals.”

RDA was developed by a committee comprising Lydia J.  Roberts, Hazel
Stiebeling, and Helen S. Mitchell during the Second World War, and the committee
was set up by the US National Academy of Sciences.

RDA of vitamin D has been established on the basis of minimum exposure to the
sun. The daily requirement of vitamin D for adults, pregnant women, and children
is given in Table 11.2.

A bsorption, Transport, and Storage of Vitamin D
Site and Process of Absorption

• Vitamin D is absorbed from the mucosa of the duodenum and jejunum.
• Bile salts are necessary for absorption of vitamin D.

Transport of Vitamin D

• It is transported from enterocytes in form of chylomicrons.
• It enters lacteals and blood circulation along with chylomicrons.
• In plasma, vitamin D binds to α2 globulin. It is transported to body tissues in

bound form.

Storage

• It is stored in the liver.

Biosynthesis of Calcitriol
Calcitriol is 1,25-dihydroxycholecalciferol. It is physiologically active form of vita-
min D. It is called “hormone.” Its biosynthesis involves the following steps:

1. Synthesis of Cholecalciferol in the Skin
• The 7-dehydrocholesterol is present in the epidermis (stratum basale and
stratum spinosum) of the human skin. It is considered as provitamin D3.

296 11 Vitamins

• The UVB radiation can easily penetrate the epidermis. The

7-­dehydrocholesterol absorbs UVB radiation (290–315 nm wave length).

It undergoes photolysis.
• Photolysis of 7-dehydrocholesterol involves two steps:

–– Opening of B-ring: Absorption of light photon results in activation of

double bonds in B-ring of 7-dehydrocholesterol. There is a cleavage of

bond between C9 and C10. The B-ring at C9–C10 position opens up. It leads
to the formation of 9,10-secosteroid called “previtamin D3.”
–– Antarafacial sigmatropic hydrogen shifting: The previtamin D3 is a pre-
cursor to vitamin D3, and it exists in two isomeric forms as “cis previtamin
D3” and “trans previtamin D3.” The former isomeric form is thermody-
namically unstable. It undergoes spontaneous antarafacial sigmatropic

shifting of h­ ydrogen atom from C19 to C9 atom. It is an intramolecular
rearrangement of atoms. It results in the formation of thermodynamically
stable 9,10-secosteroid, called “vitamin D3.”

Previtamin D3 Vitamin D3 (Cholecalciferol)

• From the epidermis, vitamin D3 passes into capillaries in the dermal layer of
the skin. Ultimately, it is transported to the liver.

2. Synthesis of 25-Hydroxycholecalciferol in the Liver

• Within the liver, cholecalciferol undergoes hydroxylation at C25 position. The
reaction is catalyzed by “25-hydroxylase enzyme” as in Fig. 11.4.

• 25-hydroxylase enzyme: It is a liver microsomal enzyme. It requires

NADPH, Mg++, and cytochrome P450.

• Cholecalciferol is converted into 25-hydroxycholecalciferol (25-HCC). It is

the chief storage form of vitamin D3 in the liver.
• A small amount of 25-hydroxycholecalciferol enters blood circulation. In

plasma, it binds with “vitamin D binding protein” and circulated to body tis-

sues. It reaches kidneys.
3 . Synthesis of 1,25-Dihydroxycholecalciferol in Kidneys

• Within kidneys, the 25-hydroxycholecalciferol undergoes second hydroxyl-

ation at C1 position. The reaction is catalyzed by “1-alpha-hydroxylase
enzyme.” The enzyme is present in mitochondria of proximal convoluted

tubules as in Fig. 11.4.

• The enzyme requires ferredoxin reductase, cytochrome P450, and Mg++ ions.

• There is formation of 1,25-dihydroxycholecalciferol (1,25-DHCC).

• It is also called “calcitriol.” It is due to the presence of three hydroxyl groups

at C1, C3, and C25 positions in the molecule, and it regulates calcium
metabolism.

• Calcitriol acts analogous to steroid hormone.
Calcitriol is considered as “hormone,” while vitamin D3 (cholecalciferol) is
considered as “prohormone.”
Rationale: Following characteristics are put forward to justify above

statement:

• Cholecalciferol (vitamin D3) is produced in the skin on exposure to UVB light
of the sun.

11.4  Fat Soluble Vitamins 297

• Calcitriol is synthesized in kidneys on enzymatic hydroxylation of
cholecalciferol.

• Calcitriol acts like steroid hormone. It acts on nuclear receptor.
• Like hormones, site of calcitriol synthesis is different from its site of action.
• Calcitriol acts on specific target tissues like hormones. It acts on the intestine,

bones, and kidneys.
• Synthesis of calcitriol is regulated by feedback inhibition.
• Calcitriol regulates calcium metabolism in association with parathyroid hor-

mone and calcitonin.

F unctions of Vitamin D (Calcitriol)
Calcitriol acts on the intestine, bones, and kidneys in the following ways:

Effect of Calcitriol on the Intestine

• Calcitriol enhances intestinal absorption of calcium and phosphate. It acts
like a hormone as described below:
–– Calcitriol binds to nuclear receptor present in the cytosol of intestinal cells.
–– It forms calcitriol-receptor complex. This complex activates genes and regu-
lates transcription of m-RNA.
–– Calcitriol controls synthesis of “calcium binding protein” (CBP).
–– Calcium binding protein enhances uptake of calcium through intestinal cells.

• Calcitriol promotes degradation of “lithocholic acid” (LCA) in the intestine and
enhances its excretion. Lithocholic acid is a toxic bile acid that is produced in the
colon by bacterial action on chenodeoxycholic acid.

Effect of Calcitriol on Bones

• Calcitriol increases deposition of calcium and phosphate in bones.
• Calcitriol stimulates osteoblastic activity.
• It enhances hydroxyapatite content of bones.
• It is necessary for normal growth.

Effect of Calcitriol on Kidneys

• Calcitriol minimizes excretion of calcium and phosphate by renal tubules.
• It increases reabsorption of calcium and phosphate by renal tubules.

Effect of Calcitriol on Immunity

• Calcitriol acts on vitamin D receptor (VDR) which is located in immune cells.
• Calcitriol modulates innate immunity.

298 11 Vitamins

Effect of Calcitriol on Cancer Cells

• Calcitriol inhibits synthesis of hypoxia-inducible factor-1 and vascular endothe-
lial growth factor in cancer cells.

• Calcitriol inhibits angiogenesis in cancer cells. It is the formation of new blood
vessels in cancer cells. Calcitriol inhibits cancer cell proliferation.

Deficiency Disorders of Vitamin D
Deficiency of vitamin D is not common in population as it is synthesized by the skin
on exposure to sunlight. However, the following predisposing factors are responsi-
ble for its deficiency in children and adults:

1. Inhabitation in damp and congested places devoid of direct sunlight
2. Intake of diet poor in vitamin D
3. Chronic alcoholism
4 . Renal and liver disorders

Vitamin D deficiency is manifested in following disorders:

Rickets
Rickets is a clinical condition characterized by skeletal deformities in bones of
growing children owing to impaired mineralization of bones.

The word “rickets” is derived from the Greek word “rachitis” which means
“in the spine.”

Clinical Features
Deficiency of calcitriol affects activity of osteoblasts. It is involved in the defec-
tive vascularization and incomplete mineralization of bones of growing children.
This condition affects bones before the closure of epiphyseal plates. Rickets has
following features:

• Bow Legs
–– Softening of ends of long bones
–– Softening of shaft of long bones due to defective mineralization
–– It causes long bone bending and bow legs

• Knock Knees
–– The condition is found in children between 2 and 5 years of age group.
–– In an upright standing position, the knees of both sides touch each other and
the feet are wide apart.
–– The knees and ankles are swollen.

• Hot Cross Bun Appearance of Fontanelles
–– Fontanelles are the membranous gaps between the cranial bones in infant
skull. They undergo ossification between 2 months and 24 months in normal
conditions.
–– In rickets, fontanelles do not calcify normally and remain open. They appear
like hot cross bun.

11.4  Fat Soluble Vitamins 299

• Pigeon Breast
–– Pigeon chest is also called “pectus carinatum.” It is a deformity in the chest
bones.
–– It is characterized by outward positioning (protrusion) of sternum and ribs in
the chest.
–– It is due to excessive deposition of non-calcified organic bone matrix (oste-
oid) on the sternum and ribs.
–– Patients have difficulty in breathing.

• Rachitic Rosary
–– Rachitic rosary is appearance of beads over ribs.
–– It is characterized by the presence of nodules at costochondral joints in the rib
cage.
–– It is due to hypomineralization and excessive osteoid tissues over costochon-
dral joints.

Osteomalacia
Osteomalacia is the rickets in adults. It is a clinical condition characterized by soft-
ening of bones in adults owing to impaired mineralization of bones.

Diminished Innate Immunity

• Deficiency of calcitriol diminishes innate immunity.
• Calcitriol acts through vitamin D receptor on antigen-presenting cells of immune

system. Its deficiency inhibits differentiation and maturation of antigen-p­ resenting
cells (APC). Immature APCs are unable to stimulate activation of T cells and B cells.
• Deficiency of calcitriol also suppresses secretion of interleukin-10 and interleu-
kin-1­ 2 which are necessary for activation of lymphocytes.

The word “osteomalacia” is derived from the Greek words “osteo” which
means “bone” and “malacia” which means “softness.”

Clinical Features

• Osteomalacia is associated with defective mineralization of bones due to decrease
in vitamin D, calcium, and phosphate levels.

• Initially, bone pain starts in lumbar region of the body. The involved bones
become sensitive to touch.

• Tenderness of bones in arms, legs, and spine.
• Proximal muscles of pelvic girdle become weak. It affects position of the body

during walking (gait). Patient assumes “waddling gait.”
• In progressive condition, bones become susceptible to pathological fracture.

Renal Osteodystrophy
Renal osteodystrophy is a condition characterized by degeneration of renal paren-
chyma and defective synthesis of calcitriol.

300 11 Vitamins

Clinical Features

• Chronic renal failure
• Bone pain and tenderness of bones
• Muscle weakness and tingling sensation over extremities
• Osteomalacia
• Hypocalcemia

Hypervitaminosis D
It is the toxicity associated with consumption of excessive amount of vitamin D. The
toxicity is induced by sustained hypercalcemia and hyperphosphatemia. Important
clinical manifestations are as follows:

• Nausea and vomiting and abdominal pain
• Constipation
• Loss of appetite, lethargy, and loss of weight
• Increased thirst and polyuria
• Increased probability of urinary lithiasis (kidney stone)
• Calcification of arteries, muscles, pulmonary bronchi, and gut mucosa
• Renal failure

Vitamin D3 (Calcitriol) and Health of Periodontal Tissues

Mineral Homeostasis

• Calcitriol influences calcium and phosphate homeostasis in the body. It regulates
mineralization of mandible and maxilla. So it influences bone density of jaw
bones.

• Calcitriol is necessary for normal bone remodeling.
• Calcitriol reduces alveolar bone resorption.
• In a clinical trial through randomization by (Garcia et al. 2011), it was observed

that supplementation of calcium and vitamin D improved severity of periodontal
infection.

Antibacterial Effect of Calcitriol

• Calcitriol acts through its vitamin D receptors located on immune cells. It inhib-
its release of pro-inflammatory cytokines through immune cells.

• Calcitriol also stimulates macrophages to release peptides. These peptides have
antibacterial effect on the microbes responsible for periodontal disease.

• Therefore, calcitriol minimizes incidence of inflammation and infection in peri-
odontal tissue.

11.4  Fat Soluble Vitamins 301

11.4.3  Vitamin E

Vitamin E is a group of naturally occurring fat soluble organic compounds
with antioxidant activity.

Vitamin E includes:

1 . Tocopherols (T)
2 . Tocotrienols (T3)

They are collectively called as tocochromanol.
The word “tocopherol” is derived from the Greek words “tokos” which means
“birth,” “pherein” which means “to carry,”
and “ol” which means “alcohol.”

H istory
• Vitamin E is called “anti-sterility vitamin.”
• In 1922, the physician Dr. Evans and his assistant, Katherine S Bishop, recog-

nized the importance of fat soluble factor in diet for normal reproduction in rats
and named it as vitamin E.
• In 1936, alpha-tocopherol was isolated from wheat germ oil by Evans and his
coworkers.
• In 1938, the structure of vitamin E was described by Erhard Fernholz.
• In 1938, Paul Karrer synthesized vitamin E.

C hemical Structure
1. Forms of Vitamin E

• Vitamin E exists in nature in eight homologues.

• Four homologues belong to tocopherols and another four belong to

tocotrienols.

• Tocopherol homologues exist as alpha-tocopherol, beta-tocopherol, gamma-­

tocopherol, and delta-tocopherol.

• Tocotrienol homologues exist as alpha-tocotrienol, beta-tocotrienol, gamma-­

tocotrienol, and delta-tocotrienol.
2 . Structure

• All compounds with vitamin E activity are synthesized by plants from homo-

gentisic acid.

• They have a common structural characteristic “6-chromanol ring.” It is a ben-

zodihydropyran as in Fig. 11.5.

• All vitamin E homologues are derived from 6-chromanol.

• Hydroxyl group is attached to C6 position at chromanol ring. It provides
hydrogen atom to free radicals. It is responsible for the antioxidant property

of vitamin E.

• A hydrophobic 16 carbon side chain is attached at C2 position of chromanol
ring.

302 11 Vitamins

5th methyl
group

CH3 CH2

C 4

5

HO C 3 CH2

6

C7 8 O 2 C C(HC3H2)3 CH (CH2)3 CH (CH2)3 C CH3
H3C CH3 CH3 CH3
C 1

7th methyl CH3
group
8–methyl
group
[5,7,8 – Trimethyltocol]

∝ – Tocopherol

Fig. 11.5  Alpha Tocopherol

• Tocopherols differ from tocotrienols in the structure of side chain. In tocoph-
erol, side chain (phytyl chain) is saturated, while in tocotrienols, side chain is
unsaturated with three double bonds at C3′, C7′, and C11′ positions.

• All vitamin E homologues differ by the number and position of methyl groups
which are attached to 6-chromanol ring.

• Examples:
–– Alpha homologues (tocopherol and tocotrienols) contain three (5, 7, 8)
methyl groups.
–– Beta homologues (tocopherol and tocotrienols) contain two (5, 8) methyl
groups.
–– Gamma homologues (tocopherol and tocotrienols) contain two (7, 8)
methyl groups.
–– Delta homologues (tocopherol and tocotrienols) contain one (8′) methyl group.

• All chemical forms of vitamin E are amphipathic molecules. The side chain is
lipophilic in nature, whereas the chromane ring is hydrophilic in nature.

• Each tocopherol has three chiral centers. First chiral center is positioned at
C2 in chromanol ring, and second and third chiral centers are positioned at C4′
and C8′ in side chain. Therefore, each tocopherol has (23) eight stereoisomers.

• Naturally occurring alpha-tocopherol exists in single stereoisomeric form
represented as RRR-α-tocopherol. Synthetic alpha-tocopherol is a race-
mic mixture (equal proportion) of eight stereoisomers and represented as
all-racemic or all-rac.

• Tocotrienols have single chiral center at C2 position.

11.4  Fat Soluble Vitamins 303

• Biological activity and antioxidant property of all chemical forms of vitamin
E are not identical.

• Gamma-tocopherol: It is the most abundant form of vitamin E in human
diet. Its biological activity is 1/10th of alpha-tocopherol.

• Alpha-tocopherol: It has the highest antioxidant activity among humans. Its
reactivity toward reactive oxygen species is higher than other tocopherols and
polyunsaturated fatty acids.

Dietary Sources of Vitamin E
1 . Animal Sources

• Meat, milk, butter, and egg yolk
2 . Plant Sources

• Tocopherols are abundant in cotton seed oil, sunflower oil, peanut oil, corn,
and soya oils.

• Wheat germ oil has the highest concentration of alpha-tocopherol. About 1
tablespoon full of oil has around 20 mg of vitamin E.

• Corn oil and soya bean oil are good sources of gamma-tocopherol.
• Tocotrienols are found in rice bran, barley, and palm oil.

R ecommended Dietary Allowance of Vitamin E
Recommended Dietary Allowance (RDA) is “average quantity of a nutrient which
is sufficient to fulfill the physiological needs of almost all (97.5%) healthy
individuals.”

RDA was developed by a committee comprising Lydia J. Roberts, Hazel Stiebeling,
and Helen S. Mitchell during the Second World War, and the committee was set up by
the US National Academy of Sciences. Table 11.3 shows RDA of vitamin E.

A bsorption, Transport, and Storage of Vitamin E
Site and Process of Absorption

• Food contains free and esterified forms of tocopherols. Alpha-tocopherol is the
most active chemical form of vitamin E.

• Tocopherols are incorporated into mixed micelles. Esterified forms are hydro-
lyzed by pancreatic lipase. Bile salts are necessary for absorption of
tocopherols.

• Free tocopherols are absorbed in the mucosa of the duodenum and jejunum. The
absorption of tocopherols occurs by passive diffusion along with dietary lipids.

• Inside enterocytes, tocopherols are incorporated into chylomicrons.
• Tocopherols leave enterocytes and enter into lacteals and lymph vessels.
• Tocopherols along with chylomicrons pass into systemic circulation.

Table 11.3  RDA of vitamin E Age group RDA for vitamin E (IU)

Children 10–15 IU
Adults 20–28 IU

1 IU = 0.67 mg of alpha-d-tocopherol

304 11 Vitamins

Transport of Vitamin E in Plasma

• Within blood circulation, chylomicrons are catabolized by lipoprotein lipase
enzyme. Fatty acids and alpha-tocopherol are released. Alpha-tocopherol is dis-
tributed to the muscles, adipose tissues, cardiac muscles, and brain tissues.

• Dilapidated chylomicrons containing alpha-tocopherol are taken up by the liver.
• Within the liver, triglycerides and tocopherol are secreted into VLDL, and they

are exported from the liver into circulation.
• Within blood circulation, a few VLDL are catabolized by lipoprotein lipase and

are converted into LDL. The VLDL remnants with tocopherol are taken up by the
liver. Tocopherol is again secreted by the liver into IDL.
• Alpha-tocopherol has lipophilic nature. It is distributed along with lipoproteins
from the liver to muscle tissues and adipose tissues for storage.

Storage

• Alpha-tocopherol is stored in parenchymal cells of the liver. Trace amount of
alpha-tocopherol is also stored in stellate and Kupffer cells of the liver.

• It is also stored in skeletal muscles and adipose tissues.

Functions of Vitamin E
Antioxidant Function

• Vitamin E inhibits the synthesis of free radicals in the body.
• Predominantly, vitamin E scavenges free radicals from body tissues as in

Fig. 11.6. It acts by the following mechanisms:
–– The chromanol ring of tocopherol furnishes its hydrogen that neutralizes

the free radicals.
–– The “chromanol ring” of tocopherol is oxidized into “quinone ring.”
• Vitamin E prevents lipid peroxidation.
–– It protects integrity of cell membrane of body cells.
–– It protects integrity of mitochondrial membrane. Hence, it maintains oxida-

tive phosphorylation in the mitochondria.
–– It protects integrity of membranes of erythrocytes and prevents hemolysis.
• Vitamin E protects coenzyme A (CoA.SH) from free radicals by preventing
oxidation of sulfhydryl group.
• Vitamin E prevents oxidation of retinol.

Cardioprotective Activity
Vitamin E is helpful in prevention of coronary artery disease (CAD). It exerts its
cardioprotective function through following activities:

• Vitamin E inhibits synthesis of “vascular cell adhesion molecule” in endothelial
cells. So it inhibits “platelet adhesion” to vascular endothelium, and it also pre-
vents “platelet aggregation.”

11.4  Fat Soluble Vitamins VITAMIN E 305
Free radical
Peroxidation Phospholipid
of molecule

PUFA in membrane

Cell membrane

PUFA – PEROXL
FREE RADICAL

CH3 O Breaks
Peroxidation
H3C CH3 Chain Reaction
O
HO PUFA in
CH3 Cell membrane

∝ – Tocopherol Ascorbate [Reduced]
or

Glatathione [G ∼ SG]

CH3

O Ascorbate [Oxidized]
or
Oxidized - tocopherol
[Quinone] Glatathione [GS ∼ SG]

Fig. 11.6  Mechanism of antioxidant function of Vitamin E

306 11 Vitamins

• Vitamin E increases synthesis of prostacyclin and nitric oxide from vascular
endothelial cells. These are potent vasodilators. They improve coronary artery
stenosis.

• Tocopherols and tocotrienols have been found to inhibit enzyme, 3-hydroxy-­
3-methylglutaryl-CoA (3HMG-CoA) reductase. This enzyme is responsible
for biosynthesis of cholesterol by the liver. Therefore, vitamin E prevents
hypercholesterolemia.

• Overall, vitamin E prevents coronary artery stenosis and atherosclerosis which
are implicated in initiation and progression of CAD.

Anticarcinogenic Property
It has been demonstrated that gamma- and delta-tocopherols have anticariogenic
property.

• Gamma-tocopherol can inhibit proliferation of cancer cells in culture by
different mechanisms:
–– Gamma-tocopherol removes free radicals which can cause mutation of
genetic material of cell (DNA).
–– It inhibits synthesis of “cyclins” which is responsible for proliferation of
cancer cells.
–– It induces apoptosis in cancer cells.

• Gamma- and delta-tocopherols have better anticariogenic property than
alpha-tocopherol.

• Scientists have reported anticariogenic activity of delta- and gamma-tocopherols
in lung, colon, prostate, and breast malignancy.

However, various studies including case control and randomized trials con-
cerning the anticariogenic property of vitamin E have yielded mixed outcome.

Reproductive Activity of Vitamin E
Vitamin E has been found to be essential for normal health of seminiferous epithe-
lium in rats. Its deficiency has been associated with irreversible changes in the epi-
thelium. It could lead to sterility in male rats.

In female rats, vitamin E is necessary for normal growth of fetus in rat models.

Role in Immunity
Vitamin E is an immune modulator. It stimulates cell-mediated immunity and
humoral immunity in humans.

• It enhances phagocytosis by lymphocytes and macrophages in bacterial
infection.

• It stimulates differentiation of immature T cells.

It has been found in randomized human trial that vitamin E supplementa-
tion improved the cell-mediated immunity in healthy old-aged persons.

11.4  Fat Soluble Vitamins 307

D eficiency Disorders of Vitamin E
Vitamin E deficiency is responsible for many deficiency disorders in the body.

• Hemolysis of erythrocytes increases in deficiency of vitamin E. It is due to lytic
action of peroxides on the cell membrane of erythrocytes.

• Vitamin E deficiency is related to hemolytic anemia.
• Male rats suffer from sterility.
• Increased prevalence of fetus resorption in female rats.
• Hepatic necrosis.

–– Due to deficiency of amino acid cysteine in diet, the liver undergoes necrosis.
Vitamin E and selenium have protective role in hepatic necrosis.

• Muscular dystrophy.
–– Deficiency of vitamin E is related to muscular dystrophy (skeletal muscle
weakness and irreversible damage).
–– Its deficiency is responsible for increased lipid peroxidation in muscles and
increased synthesis of peroxides. It results in higher hydrolase activity which
damage muscles.

• Ataxia and vitamin E disorder (AVED).
–– It’s an inherited neurological degenerative disorder in which individuals have
deficiency of vitamin E in the body. It involves neurological manifestation.
Persons lack coordination in skeletal muscles, and their walking is affected
(ataxia).

Therapeutic Value of Vitamin E

• Vitamin E has preventive and curative role in nocturnal muscle cramps (painful
condition that occurs in the leg, thigh, or feet).

• Vitamin E improves intermittent claudication (cramp in calf muscle on walking
due to occluding blood vessels).

• Vitamin E is helpful in prevention of atherosclerosis. It is due to its potent anti-
oxidant and anti-inflammatory activities.

• Vitamin E also has a therapeutic role as anti-sterility vitamin.
• AVED improves with supplementation of vitamin E.

11.4.4  V itamin K

Vitamin K belongs to a group of naturally occurring fat soluble organic com-
pounds possessing a common chemistry as “2-methyl-1,4-naphthoquinone.”

The generic name of vitamin K is derived from the German word “koagula-
tion.” It is due to its essentiality for synthesis of coagulation factors by the liver and
regulation of clotting.

308 11 Vitamins

History
• Henrik Dam and Schonleyder discovered an “antihemorrhagic factor” in chicks

around 1929  in Denmark. They observed that chicks suffered from prolonged
bleeding time after feeding with cholesterol deficient diet.
• H. Dam isolated vitamin K1 from alfalfa sprouts in 1939.
• E. A. Doisy isolated vitamin K2 in 1939.
• Dam and Doisy were awarded Nobel Prize in 1939.

Chemical Structure
1. Forms of Vitamin

Vitamin K occurs in nature in two forms which are derivative of naphthoqui-
none. The two forms are as follows:
• Vitamin K1

–– It is called “phylloquinone.” It is found in chloroplasts in leaves of plants.
–– Phylloquinone contains a methyl naphthoquinone ring. A phytyl chain

(partially saturated poly-isoprenoid alcohol) is attached to the ring.
–– Vitamin K1 is chemically 2-methyl, 3-phytyl, 1,4-naphthoquinone as in

Figs. 11.7 and 11.10.
–– It is derived from leaves of alfalfa.
–– It is also called “phytonadione.”
–– It is a yellow-colored thick oil.
• Vitamin K2
–– It is called “menaquinone.” It is synthesized by bacteria in the intestine.
–– Menaquinone contains a methyl naphthoquinone ring to which a phytyl

chain (unsaturated poly-isoprenoid chain with different carbon length) is
attached. The number of isoprene units may vary from 4 to 13 and vitamin
K2 is called as menaquinone-n in Fig. 11.8. For example, menaquinone-7
has seven isoprenoid units containing 35 carbons in side chain and desig-
nated as vitamin K2 (35).
–– Chemically, it is 2-methyl-3-difarnesyl-1,4-naphthoquinone.
–– It was isolated from decayed fish meal.
–– It is a yellow-colored thick oil.
• Vitamin K3
–– It occurs as synthetic analog of vitamin K, and it represents its third form.
It is called “menadione.”

O
CH3

CH3 CH2 CH3
CH2.CH C CH2 (CH2 CH CH2)3 H

O

Fig. 11.7  Phylloquinone (Vitamin K1)

11.4  Fat Soluble Vitamins 309

Fig. 11.8 Menaquinone O
(Vitamin K2) CH3

(CH2–CH– C–CH2)6– H

CH3
O

Fig. 11.9 Menadione O
(Vitamin K3) CH3

O

–– Chemically, it is 2-methyl-1,4-naphthoquinone. It lacks the side chain
as in Fig. 11.9.

–– Vitamin K3 is water soluble.
–– It has higher activity than its natural counter parts.
–– It is used as parenterally in management of hypoprothrombinemia.
–– Vitamin K3 exists into another two forms called “menadiol” and

“menadioldiacetate.”

D ietary Sources of Vitamin K
Vitamin K1 is present mainly in green leafy vegetables. Predominant sources are
spinach, alfalfa, cabbage, cauliflower, tomatoes, kale, turnip, and soya bean.

Vitamin K2 is mainly produced by intestinal bacteria in humans. The long-­
chain menaquinones are primarily synthesized by gut bacteria in humans.

Chief menaquinones synthesized by intestinal bacteria are as follows:

• MK-10-13 are synthesized by Bacteroides.
• MK-8 by Enterobacter.
• MK-7 by Veillonella.
• MK-6 by Eubacterium lentum.

Vitamin K3 is man-made. It is a structural analog of vitamin K1 and K2
(Fig. 11.10).

310 11 Vitamins

Fig. 11.10  2–Methyl, 3–Hydroxy, O CH3
1, 4–Naphthoquinone 2C

4 C CH3
3

O

Table 11.4  RDA of Age group RDA for vitamin K (IU)
vitamin K
Children 60 mcg
Adult males 100 mcg
Adult females 90 mcg

Source: (NIH 2017)
Daily requirement of vitamin K is between 50 and 100 mg
This amount of vitamin K (K1) is available in daily food.
The intestinal bacteria synthesize vitamin K2
Under normal conditions, deficiency of vitamin K has not
been reported among healthy persons

Recommended Dietary Allowance of Vitamin K
Table 11.4 shows RDA of vitamin K as follows:

A bsorption, Transport, and Storage of Vitamin K
Site and Process of Absorption

• Dietary vitamin K1 is incorporated into mixed micelles and is absorbed by
mucosa of small intestine. Within enterocytes, vitamin is incorporated into chy-
lomicrons. It is exported from enterocytes along with chylomicrons into lacteals
and transported into lymph vessels.

• Vitamin with chylomicrons enters systemic circulation through thoracic duct.
• The presence of bile salts is necessary for its absorption.
• Menaquinones are absorbed from gut mucosa and enter hepatic portal

circulation.

Transport of Vitamin K

• Phylloquinone and menaquinones are transported in the plasma along with
lipoproteins.

• Phylloquinone is the chief circulating form of vitamin K which is derived
from diet.

• Menaquinones, mainly MK-7, are present in the plasma in comparatively lower
concentration, and it is mainly derived from intestinal flora.

11.4  Fat Soluble Vitamins 311

Storage

• Menaquinones, predominantly MK-7, are stored in the liver in large amount.
• Phylloquinone is stored in the liver in a negligible amount.
• Hepatic storage of phylloquinone is highly labile. It undergoes depletion within

2–3 days after the stoppage of dietary intake.
• Phylloquinone and menaquinones are also stored in small amounts in the bones

and adipose tissues.

Functions of Vitamin K
Role of Vitamin K in Carboxylation of Blood Clotting Factors

• The liver synthesizes blood clotting factors. The factors II, VII, IX, and X are

synthesized in inactive forms.

• These clotting factors undergo posttranslational modification with the help of

vitamin K as described below:
–– Vitamin K is changed into hydroquinone form in liver microsomes. The

reaction is catalyzed dehydrogenase enzyme in the presence of NADPH.
–– Hydroquinone acts as coenzyme for carboxylase enzyme.
–– Carboxylase brings about carboxylation of glutamic acid residue present

in clotting factors. Additional COOH group is incorporated at gamma carbon

position in glutamic acid.
–– It results in the formation of γ-carboxyglutamate.
–– In prothrombin, initial ten glutamate residues undergo carboxylation to form

γ-carboxyglutamate residues which combine with calcium ions to form
prothrombin-calcium complex. This complex attaches to phospholipids on

surfaces of platelets.
–– It ensures rapid conversion of prothrombin to thrombin and helps in blood

clotting.
–– Drug like dicumarol is an antagonist of vitamin K. The synthetic analog

like warfarin inhibits formation of γ-carboxyglutamate. They act as
anticoagulants.

Role in Oxidative Phosphorylation in Mitochondria

• Vitamin K acts as coenzyme in oxidative phosphorylation in the mitochondria.

D eficiency Disorders of Vitamin K
Deficiency of vitamin K is unusual owing to its synthesis by intestinal bacteria as
well as intake of wide varieties of vegetables and oils. However, its deficiency
occurs in following conditions:

Prolonged Oral Administration of Broad-Spectrum Antibiotics

• Oral administration of broad-spectrum antibiotics like penicillin and cephalo-
sporin for prolonged period can inhibit growth of intestinal flora.

• It results in deficiency of menaquinones.

312 11 Vitamins

Malabsorption Syndrome

• Malabsorption is an inflammatory condition of intestinal mucosa.
• Absorption of dietary vitamin K is impaired.
• It causes deficiency of vitamin K.

H emorrhagic Disorder in Infants
Deficiency of vitamin K is seen in malnourished infants.

It is attributed to following causes:

• Low count of colonic bacteria in infants.
• Distribution of vitamin K from the body of mother to fetus is limited.
• Breast milk contains low concentration of vitamin K.

Deficiency of vitamin K results in hypoprothrombinemia (fall in concentra-
tion of plasma prothrombin). Thus, bleeding time is prolonged.

Hemorrhagic disorder in infants can be of three types based on its onset:

1 . Early onset
• Bleeding manifests within 24 h of birth of infants.

2 . Classic
• Bleeding manifests within first week after birth of infants.

3. Late onset
• Bleeding manifests within 6 months after birth of infants.

Minor injury causes prolonged capillary bleeding. It is associated with discolor-
ation of the skin which is revealed during physical examination of infants.

11.5 Water Soluble Vitamins

11.5.1 V itamin C

Vitamin C is a naturally occurring water soluble organic compound having
structural similarity to 6-C monosaccharide (l-glucose) with potent antioxi-
dant activity.

Vitamin C is called “ascorbic acid” owing to its “antiscorbutic property.”
Ascorbic is derived from the Latin word “scorbuticus” which stands for
“scurvy.”

History
• In 1490, Vasco da Gamma reported that his crew members fell sick with painful

gums and swelling of hands. They were treated with oranges and were cured.

11.5  Water Soluble Vitamins 313

• In 1600 ad, Sir Richard Hawkins, an English sea captain, reported that 10,000
sailors were severely affected by an amazing disease (scurvy) owing to dietary
deficiency. They died.

• In 1747, James Lind conducted a first trial on patients who suffered from scurvy.
He concluded that inadequate fruits and vegetables in diet was the main cause for
scurvy.

• In 1753, James Lind wrote a “Treatise on Scurvy.”
• In 1927, Zilva reported the presence of an “antiscorbutic factor” in lemon juice.
• In 1928, S. Gyorgi extracted an active ingredient from oranges and cabbage. It

was named “hexuronic acid.”
• In 1932, Waugh and King extracted vitamin C from lemon juice and crystallized it.

Chemical Structure
1. Forms of Vitamin C

• Vitamin C exists as d-ascorbic acid and l-ascorbic acid.
• d-Ascorbic acid lacks antiscorbutic activity and it is biologically

inactive.
• Vitamin C exists in nature as l-ascorbic acid as in Fig. 11.11.
2. Structure
• Its structure was established by E. L. Hirst, A. Szent-Gyorgyi, F. Michael,

and K. Kraft in 1933.
• Chemically, vitamin C (ascorbic acid) is a 2,3-enediol-l-gulonic

acid-g-lactone.
• Vitamin C has structure similar to l-glucose.
• It is an “enediol-lactone.”
• It has potent acidic property which is attributed to two hydroxyl groups

attached to C2 and C3 linked by a double bond.

Fig. 11.11  Forms of O 2H O
Vitamin C C1 Oxidation
HO C2 Reduction C1
HO C3 O O C2 O
2H O C3
H C4
HO C5 H H C4

6CH2 OH HO C5 H

L-Ascorbic acid 6CH2 OH
(Reduced state) L-Dehydroascorbic acid

(Oxidized state)

314 11 Vitamins

• Ascorbic acid can furnish two hydrogen atoms from hydroxyl groups and is
oxidized into “dehydroascorbic acid.” Ascorbic acid and dehydroascor-
bic acids are physiologically active. In the human body, ration of ascorbic
to dehydroascorbic acid is 15:1.

• Thus, it is a potent reducing agent and electron donor. It donates electrons
from double bond between C2 and C3. All biochemical activities of vita-
min C are due to its electron donation property.

• Oxidation of ascorbic acid renders it inactive. The oxidative process is
enhanced in the presence of copper and silver ions.

D ietary Sources of Vitamin C
Plant Sources

• Indian gooseberry (Phyllanthus emblica) is the richest source of vitamin
C. It contains around 400–600 mg of ascorbic acid in 100 g of gooseberry.

• Citrus fruits like orange, lemon, and tangerine.
• Fruits like strawberries, kiwi, guava, and papaya.
• Vegetables like tomato, cabbage, and spinach.

R ecommended Dietary Allowance of Vitamin C
Table 11.5 shows RDA of vitamin C as follows:

Absorption, Transport, and Storage of Vitamin C
Site and Process of Absorption

• Humans lack the enzyme “l-gulono-lactone oxidase” necessary for conversion
of glucose to ascorbic acid. Therefore, vitamin C is supplied in diet.

• Dietary vitamin C is absorbed through intestinal mucosa.

Storage

• Vitamin C is not stored in the body.

Table 11.5  RDA of vitamin C Age group RDA for vitamin C
40–50 mg (AI)
Infants (0–12 months) 15–25 mg
Preschool children
(2–5 years) 25–45 mg
Children (6–12 years) 65–75 mg
Adolescents (12–18 years) 90 mg
Adult male (>18 years) 75 mg
Adult female (>18 years) 85 mg
Pregnancy (>18 years) 120 mg
Lactation
Old aged (>50 years)

Source: (NIH 2017)
AI adequate intake

11.5  Water Soluble Vitamins 315

Transport of Vitamin

• Vitamin C is distributed widely in body tissues by blood circulation.
• Organs like the adrenal gland, pituitary gland, liver, and brain have higher con-

centration of vitamin C owing to their higher metabolic activities.
• Vitamin C can cross placental barrier. It is supplied to the fetus in adequate

concentration.

Excretion

• Vitamin C is excreted in urine as such.
• It is metabolized into oxalic acid and diketogulonic acid. These metabolites are

excreted in urine.

Functions of Vitamin C
Antioxidant Activity

• Antioxidant is an element or compound that prevents oxidation of another bio-
molecules. Vitamin C is one of the potent water soluble antioxidant vitamins.
Others are Vitamin E and Vitamin A.

• It helps to remove free radicals from body tissues.
• It prevents body tissues from oxidative stress. Prominently, lipids, proteins, and

DNA are protected by vitamin C from oxidative damage.
• Free radicals are highly involved in the pathogenesis of coronary artery disease,

colon cancer, atherosclerosis, cataract, and other degenerative disorders of the
body. Vitamin C plays a preventive role in the.

Activity in Bone Metabolism

• Ascorbic acid stimulates osteoblasts and fibroblasts. It promotes synthesis of
osteoid formation (organic bone matrix).

• Osteoid is mineralization by deposition of hydroxyapatite crystals.

Activity in Collagen Synthesis

• Procollagen (unhydroxylated collagen) is made up of triple helix. It has three
polypeptide chains in which glycine is located at every third position.

• The chains are helically coiled in a left-handed direction. They in turn coil in a
right-handed direction to form a super helix (triple helix).

• Procollagen contains predominantly glycine and proline amino acids.
• Conversion of precollagen into collagen molecule requires hydroxylation of pro-

line and lysine residues.
• Hydroxylation reaction is catalyzed by prolyl hydroxylase and lysyl hydroxylase

enzymes.

316 11 Vitamins

• Ascorbic acid acts as coenzyme along with molecular O2 and Fe+++in hydroxyl-
ation process.

• Hydroxyproline and hydroxylysine provide stability to triple helix and convert
procollagen into stable collagen.

Activity in Cholesterol Metabolism

• Ascorbic acid is involved in the synthesis of bile acids from cholesterol. In defi-
ciency of ascorbic acid, in experimental guinea pigs, it has been found that syn-
thesis of bile acid is reduced.

• Ascorbic acid deficiency impairs activity of cholesterol-7 alpha-hydroxylase
enzyme in liver microsomes.

• Ascorbic acid is also helpful in regulation of normal serum cholesterol level (less
than 200 mg/dl). Dietary deficiency of ascorbic acid has been found to be associ-
ated with hypercholesterolemia.

Activity in Corticosteroid Synthesis

• Adrenal gland possesses high level of ascorbic acid in stressful condition.
• Adrenal cortex synthesizes corticosteroid hormones under the influence of ACTH.
• Ascorbic acid plays a role in the hydroxylation process during the synthesis of

corticosteroid hormone.
• Adrenal cortex contains high concentration of ascorbic acid. During stress, its

concentration decreases.
• In conditions like acute infection, fever, and renal and liver diseases, the level of

ascorbic acid in blood circulation is decreased. This indicates role of ascorbic
acid in combating infection and disease.

Activity in Catecholamines Synthesis

• Dopamine is converted into norepinephrine through hydroxylation in adrenal
medulla.

• Hydroxylation reaction is catalyzed by dopamine hydroxylase.
• Ascorbic acid acts as coenzyme in hydroxylation process.

Activity in Carnitine Synthesis

• Carnitine is helpful in the transportation of activated fatty acid from cytosol to
inside the mitochondria.

• Carnitine is synthesized in the liver through hydroxylation of gamma-­
butyrobetaine by dioxygenase enzyme.

• Ascorbic acid acts as coenzyme in hydroxylation reaction along with
α-ketoglutarate and Fe+++.

11.5  Water Soluble Vitamins 317

Activity in Cellular Redox Reaction

• Ascorbic acid is helpful in oxidation-reduction biochemical reactions in the
body.

• It plays its role owing to reversibility of ascorbate-dehydroascorbate reaction.
• It acts as hydrogen donor in the redox reactions.

Activity in Electron Transport Chain

• Ascorbic acid is necessary for normal functioning of ETC in the mitochondria.
• It is essential for optimum activity of cytochrome oxidase enzyme, a constituent

of ETC.

Activity in Folic Acid Metabolism

• Ascorbic acid is necessary for conversion of folic acid into tetrahydrofolate by
enzyme dihydrofolate reductase.

• Ascorbic acid keeps dihydrofolate reductase in reduced form.
• Ascorbic acid and tetrahydrofolate are essential for maturation of erythrocytes.

Activity in Ferritin Synthesis

• Ascorbic acid is helpful in the synthesis of ferritin (storage form of iron in body
tissues).

• Ascorbic acid is also necessary for iron mobilization from iron store (ferritin).

Activity in Hemoglobin Metabolism

• Ascorbic acid is necessary for biodegradation of hemoglobin (released from
senile erythrocytes) into bile pigments.

• It is helpful in conversion of methemoglobin into hemoglobin.

Activity in Iron Absorption

• Plant-based dietary iron is present in ferric form.
• After intake of diet, ascorbic acid helps in the conversion of ferric form of iron

into ferrous form.
• Ascorbic acid combines with ferrous iron to form Fe-ascorbate complex. It is

water soluble.
• The complex is rapidly absorbed through intestinal mucosa.

318 11 Vitamins

Activity in Immunity Modulation

• Ascorbic acid promotes synthesis of immunoglobulins. They provide humoral
immunity.

• It stimulates phagocytic action of lymphocytes and macrophages.

Activity in Tryptophan Metabolism

• Ascorbic acid acts as cofactor in hydroxylation of tryptophan.
• Tryptophan is converted into serotonin (5-hydroxy-tryptamine). It is a mono-

amine and acts as neurotransmitter.

Activity in Tyrosine Metabolism

• Ascorbic acid acts as cofactor in hydroxylation by para-hydroxy-
phenylpyruvate.

• The hydroxylation reaction is catalyzed by p-hydroxy-phenylpyruvate
hydroxylase.

• Para-hydroxy-phenylpyruvate is converted into homogentisic acid.

D eficiency Disorders of Vitamin C
Scurvy

• Scurvy is a chronic dietary deficiency disorder of vitamin C.
• It is prevalent among malnourished children, mentally challenged population,

and malabsorption syndrome.

It is characterized by the following conditions:

• Fragility of Capillaries

Capillaries become susceptible to rupture under normal pressure. It results in
bleeding in conjunctiva, retina, joint space, subperiosteal, and subcutaneous.

• Impaired Wound Healing

Wound healing is delayed. It is due to impaired collagen synthesis.

• Swollen Gums

Gums become swollen and painful. Gums bleed on slight pressure. Teeth become
loose.

11.5  Water Soluble Vitamins 319

• Impaired Osteoid formation

Activity of osteoblasts is decreased. Deposition of organic matrix (osteoid for-
mation) is decreased. Mineralization is decreased.

Formation of collagen and new capillaries is impaired.
The bone formed is weak and it is susceptible to fracture on mild pressure.

Anemia
Deficiency of vitamin C is associated with iron-deficiency anemia. It is called
microcytic hypochromic anemia. This type of anemia is generally caused by poor
absorption of dietary iron in the absence of vitamin C.

11.5.2  V itamin B Complex

Vitamin B complex is a group of water soluble organic compound chemically
distinct from each other collectively endowed with coenzyme activity.

11.5.3  Vitamin B1 (Thiamine)

History
• In 2600 bc, the oldest Chinese medical treatise mentioned thiamine deficiency in

population.
• In 1884, K. Takaki conceptualized the disease beriberi to be caused by dietary

deficiency. He ordered to replace diet containing only rice with a diet enriched
with meat, wheat, and barley.
• In 1897, Dutch physicians named C. Eijkman and Grijns in Java induced a para-
lytic state resembling beriberi in chicken by feeding polished rice. Disease was
reversed by supplementing rice polishing in diet. He received Nobel Prize.
• In 1926, Dutch workers named as B. Jansen and W. Donald crystallized vitamin
B1 from rice bran.
• In 1935, R. R.  Williams synthesized vitamin B1 and named it as “thiamine”
owing to the presence of “thiazole” and “amino groups.”
• In 1937, Lohman and Schuster extracted thiamine pyrophosphate from yeast.

Thiamine in the literature has been described as “aneurine” (which can cure
neuritis).

Thiamine is an “antineuritic vitamin” or “anti-beriberi factor.”
Thiamine is synthesized by bacteria, fungi, and plants. Animals derive thia-
mine from diet.
Thiamine is an essential nutrient for animals.

320 11 Vitamins

Chemical Structure
1. Forms of Thiamine

Thiamine (free form)
Thiamine pyrophosphate (bound form)
• It is abbreviated as TPP.
• It is a biological active form.
• It acts as coenzyme.
2. Structure of Thiamine
• Thiamine is a sulfur-enriched water soluble vitamin.
• Chemically, it is composed of two rings:

–– Pyrimidine ring: It is 2,5-dimethyl-4-aminopyrimidine.
–– Thiazole ring: It is 4-methyl-5-hydroxyethylthiazole.
–– Thiazole contains sulfur and nitrogen atoms, and it is a heterocyclic ring

(C3H3NS) as in Fig. 11.12.
• Rings are linked with methylene bridge.
• Thiamine is the exclusive compound with Thiazole ring in nature.
• Thiamine exists in bound form as “thymine pyrophosphate.” It acts as coen-

zyme. The hydroxyl group of thiamine undergoes esterification with two
phosphate residues to form thiamine pyrophosphate.

Dietary Sources of Thiamine
1 . Animal Sources

• Animal tissues contain phosphorylated thiamine (bound from).
• Good sources are meat, egg, and liver.
• Human milk and cow milk possess thiamine. It is present in trace amount

(0.04 mg/100 ml).

Methylene
bridge

3H 3 4
N C
C CH2 N C CH3
4 NH2 HC2 CH2
5 5C
H3C C 2 CH2OH
N1 6C 1

S

Pyrimidine Thiazole
ring group

Fig. 11.12  Vitamin B1

11.5  Water Soluble Vitamins 321

2 . Plant Sources
• Plant tissues contain thiamine in free form.
• Richest source is yeast.

• Good sources are cereals. Thiamine is present in variable amounts in different

fractions of grain.
–– Bran is good source of thiamine.
–– The “aleurone layer” in the bran is the richest in thiamine.

• Whole wheat and unpolished rice are good sources of thiamine.

• Additional sources are pulses, nuts and oil seeds, and green leafy vegetables.

Fractions of Wheat Grain

1. Bran (15%). It is comprised of pericarp, testa, and aleurone.
2. Germ (2%).
3. Endosperm (83%).

Milling process removes bran (rich in fibers and vitamins) including germ frac-
tion from wheat grain leaving behind starchy endosperm.

R ecommended Dietary Allowance of Thiamine
• RDA for Adults

–– Thiamine requirement is dependent on the quantity of carbohydrate con-
sumed per day.

–– It is 0.5 mg for adults for 1000 cal/day. Thiamine requirement is between 1.0
and 1.5 mg/day for adults.

• RDA for Children
–– It varies from 0.4 mg to 1.3 mg/day from infants to adolescents age groups.

• RDA for Pregnant/Lactating Women
–– Its requirement is 1.5 mg/day.

Absorption, Transport, and Storage of Thiamine
Site and Process of Absorption

• Dietary thiamine from plant sources is present in free, whereas from animal
sources is present in bound form.

• Bound form of thiamine is hydrolyzed by pyrophosphatase enzyme in the intes-
tine, and thiamine is released.

• Thiamine is absorbed by enterocytes by the following two methods:
–– At low intraluminal concentration, thiamine is absorbed by carrier-­
mediated process which is pH-dependent and Na+ independent.
–– At high intraluminal concentration, thiamine is absorbed by passive diffusion.

• Within Enterocytes (Intracellular)
–– Enterocytes contain “thiamine pyrophosphokinase enzyme.”
–– Enzyme brings about phosphorylation of thiamine into thiamine pyro-
phosphate (TPP).

322 11 Vitamins

• TPP is transported across cells (transcellular) from mucosa to serosa.
–– TPP undergoes dephosphorylation by phosphatase enzyme.
–– Free form of thiamine is released from small intestine. It is regulated by
Na+/K+ ATPase.

Storage

• Thiamine stored in the body is highly labile. Thiamine has high turnover rate.
• Thiamine storage is around 25–30 mg.
• Dietary deficiency of thiamine results into depletion body store within 2-3 weeks.
• Tissues with high thiamine storage are the heart, liver, and kidneys.
• Tissues with low thiamine storage are the brain and skeletal tissues.

Transport of Thiamine

• In blood circulation, thiamine binds to albumin. It is distributed to tissues in
complex state with albumin.

Excretion

• Thiamine is water soluble. Thiamine and its metabolites (thiazole and pyrimi-
dine) are excreted by the kidneys.

Functions of Thiamine
Thiamine pyrophosphate functions as coenzyme in biochemical reactions.

Oxidative Decarboxylation of Pyruvate

• Oxidative decarboxylation of pyruvate is catabolized by pyruvate dehydroge-
nase complex (PDH) enzyme into acetyl CoA. It occurs in aerobic glycolysis in
cells.

• TPP acts as coenzyme.
• TPP is essential in carbohydrate metabolism and energy production.

Oxidative Decarboxylation of α-Oxoglutarate (α-Ketoglutarate)

• Oxidative decarboxylation of α-oxoglutarate is catabolized by α-oxoglutarate
dehydrogenase into succinyl CoA.

• TPP acts as coenzyme.
• TPP is essential in citric acid cycle and energy production.

Non-oxidative Decarboxylation of Pyruvate

• Non-oxidative decarboxylation of pyruvate is catabolized by pyruvate carbox-
ylase into acetaldehyde. It occurs in yeasts.

• TPP acts as coenzyme.

11.5  Water Soluble Vitamins 323

Degradation of Branched-Chain Amino Acids (BCAAs)

• Valine, leucine, and isoleucine are branched-chain amino acids (BCAAs). They
undergo transamination in cytoplasm into α-keto acids.

• The α-keto acids are transported into the mitochondria and undergo oxidative
decarboxylation by branched-chain α-keto acid dehydrogenase complex (located
on inner mitochondrial membrane) into isobutyryl-CoA, 3-methylbutanoyl-CoA
and 2-methylbutanoyl-CoA.

• TPP acts as coenzyme.

Transketolation of Ribose-5-phosphate

• Transketolation of ribose-5-phosphate is catabolized by transketolase enzyme
into sedoheptulose and glyceraldehyde.

• TPP acts as coenzyme.
• TPP is essential in HMP pathway in glucose metabolism and energy production.

Conduction of Nerve Impulse

• Acetylcholine is synthesized from choline and acetyl CoA by choline acetyl-
transferase enzyme in nerves.

• TPP acts as coenzyme.
• TPP is essential in nerve impulse conduction.

Acts as Cholinomimetic

• Cholinergic neurons in brain have high concentration of thiamine.
• Thiamine enhances the cholinergic action of acetylcholine and also acts as cho-

linomimetic in the brain.

D eficiency Disorders of Thiamine
Thiamine deficiency is caused by the following factors:

• Malnutrition
• Excessive intake of tea, coffee, and betel nuts (contain high amount of thiami-

nase enzyme)
• Chronic alcoholism
• Chronic disease like diabetes mellitus and liver cirrhosis
• AIDS

Metabolic Impairment
• Increased Plasma Pyruvate Level

324 11 Vitamins

–– Pyruvate accumulates in body tissues. Its plasma concentration is elevated
[normal value (0.7–1.5 mg/dl)].

–– In deficiency of thiamine, blood-brain barrier is compromised. Pyruvate
crosses blood-brain barrier. Brain functioning is impaired.

• Oxidative phosphorylation in the mitochondria and HMP pathway are neg-
atively affected.

• Synthesis of ATP and NADPH are reduced.
• Cellular activities are impaired.
• Thiamine deficiency impairs citric acid cycle. It has a leading role in metabolism

of carbohydrates, lipids, and amino acids.
• Its deficiency inhibits synthesis of ATP, NADPH, acetylcholine, and GABA

and hence impairs nerve conduction.

Beriberi
Beriberi is a thiamine deficiency disorder. It is prevalent in regions where people
consume polished rice as staple diet.

Beriberi is characterized by the following manifestations:

• Anorexia and loss of weight
• Nausea and vomiting
• Fatigue
• Pain in limbs
• Palpitation

It is distinguished into four categories depending on clinical manifestation:
Dry Beriberi
It is associated with peripheral nerve damage.
It is characterized by the following manifestations:

• Tingling sensation in upper and lower limbs (paresthesia)
• Pain in limbs
• Absence of deep tendon reflexes (test for normalcy of spinal cord and periph-

eral nervous system)
• Loss of motor neuron functioning in lower limbs resulting into paralysis
• Ataxia (neurological condition characterized by loss of coordination in skeletal

muscles movement). Walking difficulty and abnormal gait
• Anorexia, nausea, and vomiting
• Absence of edema

Wet Beriberi
It is associated with disorders of cardiovascular system.
It is characterized by the following manifestations:

• Increased heart rate (tachycardia)
• High systolic blood pressure

11.5  Water Soluble Vitamins 325

• Rapid and bounding pulse
• Congestive cardiac failure
• Difficulty in breathing on exertion (dyspnea)
• Edema of the feet, legs, face, and abdomen
• Anorexia, nausea, and vomiting

Infantile Beriberi
It is manifested in children between 2 and 3 years of age group.
It is characterized by the following manifestations:

• Hoarseness of voice in children.
• Anorexia, nausea, and vomiting.
• Loss of weight and child becomes marasmic.
• Tachycardia in children.
• Restlessness, lack of concentration, and poor temperament.
• Edema of legs.
• Convulsions in advanced stage of disease.

Gastrointestinal Beriberi
It is associated with manifestations of GIT:

• Accumulation of lactic acid in plasma
• Abdominal pain
• Nausea and vomiting
• Anorexia and weight loss

Wernicke’s Encephalopathy
It is a thiamine deficiency-associated neurological disorder. It is common in mal-
nourished population and alcoholics.

It is characterized by the following manifestations:

• Ophthalmoplegia [paralysis of ocular muscles (superior, inferior, medial, and
lateral recti, inferior and superior oblique muscles)] responsible for eye
movements

• Ataxia
• Confusion

Korsakoff Syndrome
It is associated with loss of neurons in the brain especially in alcoholics.

It is characterized by severe loss of memory without derangement in intellectual
capabilities: