Vitamin C (Ascorbic acid)

Vitamin C (Ascorbic acid)
Dr. Rohini C Sane
Infantile scurvy-
Bayonet-rib
syndrome

In sixteen century→10000 mariners died of a miraculous disease(scurvy).
• Lack of fresh green vegetables and fruits(especially citrus) in their diet →
no supplementation Vitamin C→ suffered from scurvy.
• James Lind(surgeon of English navy) published “ Treatise on scurvy” (1753).
• Based on Lind’s observation→ compulsory rationing of lime or lemon juice to all
crews of British Royal navy .
• British sailors carrying crates of lemons →nicknamed as Limeys.
Historical background of Ascorbic acid

“Hexuronic acid”(Ascorbic acid)
Walter Haworth (Nobel prize 1937) established the molecular structure and synthesized
ascorbic acid in the same year. Albert Szent -Gyorgyi (Nobel prize 1937) isolated ascorbic acid
and named as “Hexuronic acid”.

Dietary sources of Ascorbic acid
Dietary sources of vitamin C : citrus fruits(orange ,lemons ,lime),strawberries, Indian
Gooseberry(amla), guava ,papaya, tomatoes, potatoes(particularly skin), green vegetables
(cabbage, spinach , peas, cauliflower and broccoli), germinating seeds .Milk/dairy products,
cereals, meat and eggs are poor sources of Ascorbic acid. One can hardly depend on an
adequate intake unless a certain quantity of fresh vegetables and fruits is taken each day.

Reference nutrient intake of Ascorbic acid
Category(years of age) RDA (mg/day)
Adult male(19-70) 90
Women(19-70) 75
Pregnancy and Lactation 150
Male Infants(1-3) 15
Male Children (4-8) 40
female Infants(1-3) 45
female Children(4-8) 65
Infants up to 6 months(AI) 40
Infants up to 7-12 months(AI) 50
Recommended intravenous IV intake for adult patients receiving TPN =200 mg/day
(due to increased requirement for wound healing and antioxidant activity)

Conditions associated withincreased Dietary requirement ofAscorbic acid (vitamin C)
❖Recommended daily allowance(RDA) : 70-75 mg/day(50 ml orange juice)
❖RDA of Ascorbic acid increases (100mg/day) in:
a. Pregnancy and Lactation(150mg/day)
b. Wound healing/ fracture /trauma/burns/ulcers
c. Growth
d. Aging
e. Chronic alcoholism/smoking . Cigarette smoking can increase requirement of
Ascorbic acid (turn over) due to free radical scavenging by vitamin C.
f. Oral contraception
g. Aspirin administration(Aspirin blocks uptake of vitamin C by white blood cells.)
h. Chronic diseases/infections e.g. Tuberculosis
✓ Keep in gunny bag and eat it in grams.
? Toxic effects of Ascorbic acid →kidney/urinary stones formation.

Properties of Ascorbic acid (vitamin C): 1
Ascorbic acid :
a. A hexose(6 carbon) derivative.
b. Resembles monosaccharides in
structure.
c. Acidic property of vitamin C is due to
the enolic hydroxylic groups.
d. A strong reducing agent(presence of
double-bonded →enediol carbons) .
e. L-Ascorbic acid undergoes oxidation
to form L-dehydroascorbic acid and
this reaction is reversible.
f. L-Ascorbic acid and dehydroascorbic
acid are biologically active (anti-
ascorbutic activity).
g. D-Ascorbic acid is biologically inactive.
StructureofL-Ascorbicacid(naturallyoccurringform)

Reversible oxidation of L-ascorbic acid to L-dehydroascorbic acid
Strong reducing property of L-Ascorbic acid depends on liberation of hydrogen atom from the enediol –OH
groups ,on C2 and C3 . Ascorbic acid is being oxidized to Dehydroascorbic acid(DHA) by air, H2O2 , FeCl3,
methylene blue, ferricyanide , 2-4dichlorophenol indophenol.
The above reaction is reversible by reducing agents in vitro by H2S and in vivo by –SH compounds such as
Glutathione.
Enediol
Hydroxyl
group

Properties of Ascorbic acid (vitamin C): 2
Ascorbic acid :
f. The plasma and tissue predominantly
contain Ascorbic acid in the reduced
form.
g. Ratio of Ascorbic acid to
Dehydroascorbic acid =15: 1.
h. On hydration, Dehydroascorbic acid is
converted to 2,3-diketogulonic acid
which is biologically inactive.
i. This hydration/oxidation reaction is rapid
and spontaneous, in alkaline or neutral
solution.
j. Formation of 2,3-diketogulonic acid from
ascorbic acid is regarded as biological
inactivation.
k. Oxidation of Ascorbic acid is rapid in
presence of copper (Cu 3+ ) and silver
(Ag2+) , hence vitamin C becomes
inactive if food is prepared in copper/
silver vessels .
OxidationofAscorbicacidinpresenceofcopper/silver

D- Ascorbic acid(Biologically inactive form of Vitamin C)
D-Ascorbic acid is biologically inactive.
L-Ascorbic acid and dehydroascorbic acid are
biologically active(anti-ascorbutic activity).

Properties of Ascorbic acid (vitamin C):3
• Solubility :Water soluble vitamin
• Taste: acidic
• Physical property: White crystalline substance
• Stability :Stable in solid form and in acidic solution . It is easily destroyed by
heat ,alkali and on storage . In the process of cooking, 70% of vitamin C is lost.
• Sensitivity: to oxygen, metal ions and light
• Chemical property :Reducing substance(double bonded Enediol carbon in
structure → donor of hydrogen atom),stronger acid than acetic acid.
Absorption maximum 245nm at pH < 3.
• Confirmative test: Tilman's test(2,4 Dichlorophenol Indophenol dye)-spot test
Oxidation by atmospheric oxygen exposure
Positive Tilman’s Test Blue color of dye reappears

Tilman's test : Qualitative test for Ascorbic acid
Positive Tilman’s Test Control
20 drops of
dichlorophenol
indophenol + 10
drops Ascorbic
acid
20 drops of
Dichlorophenol
indophenol(blue dye)
+ 10 drops water
2,4 Dichlorophenol indophenol dye is
reduced to colorless form by Ascorbic acid.

Chemistry of Ascorbic Acid
-2H H2O
L- Ascorbic acid D-Dehydroascorbic Acid 2,3-Diketogulonic acid
(Active) +2H (Active) (Inactive)
(Glutathione ,H2S) Oxalic Acid
(Urinary Calculi)

Biosynthesis of Ascorbic acid(Vitamin C)
• Plants and the most of animals can synthesize
Ascorbic acid from Glucose by Uronic acid
pathway.
• However, human, higher primates ,guinea pigs
and bats cannot synthesize ascorbic acid due
to deficiency of single enzyme namely
L-Gulonolactone oxidase (lack the genes
responsible for the synthesis of this enzyme) .
• They cannot convert 2-keto-L-gulonolactone
to L- Ascorbic acid.
• Thestapledietofprimatescontainsfruitsand
vegetablesrichinascorbicacidandsothegene
deletionwillhavenodeleteriouseffectsinprimates.
Humanbeings,ofcourse,carriedoverthisgene
deletion.

Conversion of Glucose to Ascorbic acid
D-Glucose Gluconolactone Ascorbic acid
absent in the Human
L-Gluconolactone oxidase
Several steps
Plants and the most of animals possess the ability to synthesize vitamin C from D-Glucose via
lactones of D-Glucuronic and L-Glulonic acid. However, some mammals including the human
lack enzyme L-Gluconolactone oxidase, the enzyme that catalyses the formation of
2-keto-L-gulonolactone , which spontaneously tautomerizes to L-Ascorbic acid.

Biosynthesis of L-Ascorbic acid from Glucose
Glucose Galactose
Glucose-6-phosphate
Glucose-1-phosphate
Uridine diphosphate-glucose
Uridine diphosphate glucuronic acid
D-Glucuronic acid
L-Gulonic acid
L-Gulonolactone 3-keto-L-gulonate
L-Xylose
2-Keto-L-gulonolactone Xylitol
D-Xylose
L-Ascorbic acid Xylulose-5-phophate
Hexose
monophosphate
shunt
↓
↓
↓
↓
↓
↓
↓
↓
↓
L- Gulonolactone oxidase O2
HumanBody,higherprimates,
monkeys,guineapigsandbats
lackgeneforsynthesisof
L-gulonolactone oxidase.
Theycannotconvert2-keto-L--
gulonolactonetoAscorbicacid.
→nobiosynthesisvitaminC.
→DietarySupplementation of
vitaminCisneeded. H2O
Glucuronides →conjugation with
bilirubin, steroid hormones , drugs
and synthesis of GAG

Conversion of L- Glucuronic acid to vitamin C
D-Glucuronic acid
NADH+ H+
CO2 NADH+ H + NAD + NADP+ H2O
L-Xylose L-Gulonic acid Gulono lactone
NADPH +H+
NADP+ absent in human
Xylitol 2-keto-L-gulono lactone
NADP+
NADPH +H+
D-Xylulose D-Xylulose -5-P L-Ascorbic acid (Vitamin C)
ATP ADP
O2
Mg 2+
Enters HMP shunt
L- Gulonolactone oxidase

Biosynthesis of Ascorbic Acid : Uronic Acid Pathway(in lower animals)
Stapledietofprimatescontainsfruitsandvegetableswhicharerichinascorbicacid.Sogenedeletionwillhave
nodeleteriouseffectinprimates.

Metabolism of Ascorbic acid
• Oxidation of Vitamin C →Dehydroascorbic acid (active)→irreversible
spontaneous hydration →Di-keto-L-Gulonic acid (inactive). This inactivation
referred to as biological inactivation.
• 2,3-Di-keto-L-Gulonic acid (inactive)→after oxidation → oxalic acid.
• Oxidation of Ascorbic acid is rapid in presence of copper. Vitamin C is
inactivated if food is prepared in copper vessels.
• Ascorbic acid is excreted unchanged and partly as oxalic acid.
• Most of oxalates in urine are derived from Ascorbic acid and rest from Glycine
metabolism.
• Human body pool of Ascorbic acid :2g

Absorption, transport and excretion of Ascorbic Acid in the human body
▪ L-Ascorbic acid : a biologically active form , D- Ascorbic acid : is a biologically Inactive form.
▪ Ratio of L-Ascorbic acid: L- Dehydroascorbic acid= 15:1
▪ Absorption: Readily absorbed in gastro-intestinal tract, peritoneum and subcutaneous
tissue, where some of Ascorbic acid is converted to Dehydroascorbic acid(DHA) form.
▪ Transport: At physiological pH , the unchanged dehydroascorbic acid passes across cell
membranes faster than the monoanionic L-ascorbate. From maternal blood ,it can cross the
placental barrier and supplies the fetus. Secreted in milk(18 -22mg/ 600-700ml of milk).
▪ Storage : No storage in Human body.
▪ Biological Half life: 8-40 days(an average 16 days)
▪ Urinary Excretion of Ascorbic acid in human body (in 3 forms):
a. Ascorbic acid
b. 2,3-Diketo-L-Gulonic acid
c. Oxalic acid
➢Low intake of vitamin C → maximum gastrointestinal absorption→ minimum urinary
excretion , high intake ≥ 500mg → excretion of unchanged Ascorbic acid over 24hours.
▪ Benedict’s test positive in urine after the vitamin C administration, indicating vitamin C is a
strong reducing agent .
DHA that is not recycled is irreversibly de-lactonized to
2,3-Diketogulonic acid.

Mechanism of absorption and transport of Ascorbic Acid
1. Gastrointestinal absorption of Ascorbic acid : by combination of sodium-dependent active transport at its
low concentration and by simple diffusion at its high concentration.
Usual dietary intake : 70-90 % (180 mg/day) absorbed. Dietary intake >1gm/day : ≤ 50% absorbed
2.Movement of Absorbed vitamin C from gastrointestinal tract → plasma : a process of facilitated diffusion
3.Uptake of Ascorbic acid by nucleated cells : mediated by specific transporters SVCT1 and SVCT2
4.Uptake of Dehydroascorbic acid by RBC : via the facilitated-diffusion glucose transporters GLUT1,3 and 4.
1
2
3, 4 Existence of Plasma vitamin C :
ascorbate ion
intracellularly DHA
reduced to Ascorbate
Human body pool
of Ascorbic acid :2g

MechanismofUptakeofAscorbicacid(bynucleatedcells)and Dehydroascorbicacid (byErythrocytes)
UptakeofAscorbicacidbynucleatedcells:mediatedbyspecifictransportersSVCT1andSVCT2
UptakeofDehydroascorbicacidbyRBCs :thefacilitated-diffusionglucosetransportersGLUT1,3and4.
UptakeofAscorbicacidbynucleatedcells:mediatedby
specifictransportersSVCT1andSVCT2
UptakeofDehydroascorbicacidbyRBCs :the
facilitated-diffusionglucosetransportersGLUT1,3and4.

Catabolic products of Ascorbic acid
L-Ascorbic acid -2H Dehydroascorbic acid 2,3-keto-Gulonic acid
(Reduced) +2H (oxidized) - H2O oxidation
irreversible
Oxalic acid
(excreted in urine)
Other catabolic products of 2,3-Diketo-Gulonic acid which are excreted
in urine:
L-Lyxonic acid L – Threonic acid L-Xylose
+ H2O

Tissue concentration of Ascorbic acid
▪ Normal level of Ascorbic acid in plasma : 0.4 to 1.5 mg/100ml (25-85μmols/L)
▪ Normal level of Ascorbic acid in WBC (leucocytes) : 25 mg/100ml WBC
▪ Low levels of Ascorbic acid in plasma: women taking contraceptive pills and in chronic
alcoholics.
▪ High levels of Ascorbic acid : in plasma locally during wound healing (Vitamin C essential
for wound healing), in the adrenals gland, pituitary gland , corpus luteum and retina (20-30
times higher than in plasma).
• Local concentration roughly parallels the metabolic activity found in descending order as
follows:
Pituitary gland >adrenal cortex > corpus luteum > liver > brain > gonads > thymus > spleen >
kidney > heart > skeletal muscles.
• It is also secreted in milk in small quantity(18 -22mg/ 600-700ml of milk).
• The vitamin C exist in body in the reduced form ,with reversible equilibrium with relatively
small amount of dehydroascorbic acid (oxidized form).
Total body pool of Human male (physiological) =1.5 -2 g
Total body pool of Human male (Scurvy)= 300mg

Laboratory assessment of Ascorbic acid status
❖Laboratory assessment of Ascorbic acid status is made by direct measurement of
plasma , urine and tissue concentration of Ascorbic acid , Total vitamin C(Ascorbic
acid + Dehydroascorbic acid) or (rarely) metabolites.
❖Plasma ascorbate concentration : is a reliable indicator of ascorbic intake.
❖Leucocyte Ascorbic acid measurement :
• Better indicator of body stores than plasma ascorbate
• Not widely adopted because:
1. Large sample volume requirement
2. The influence of fluctuating leucocyte numbers
3. Difficulty of automating the analysis
4. Difficulty of the analysis
➢Urinary excretion and RBC concentration : not specific and therefore not useful
indices of ascorbic acid status.
➢Urinary levels of Ascorbic acid : clinical diagnosis of scurvy following a load test.

Quantitative analysis of Ascorbic acid:1
❖Preanalytical treatment of plasma sample soon after phlebotomy :
• Need for Preanalytical treatment of plasma sample : As Ascorbic acid is
readily oxidized by dissolved oxygen at neutral pH.
Treatment : plasma sample + a metal-chelating and protein precipitating acid
(e.g. metaphosphoric acid)
• Storage :Treated plasma sample stored at -800C till analytical procedures .
❖Bio-assays : Guineapig is the test animal of choice .
❖Photometric (chemical) method : measurement of rate or extent of reduction
of certain oxidizing agents (dye). e.g. 2,4 Dichlorophenol indophenol dye is
commonly used and is reduced to colorless form.
❖Colorimetricassay:measurementofredcoloredcomplex(bis-hydrazone)formedby
couplingofdehydroascorbicacidwith2,4Dinitrophenylhydrazine(DNPH)andtreatment
withconcentratedH2SO4.

Quantitative analysis of Ascorbic acid:2
❖Fluorometric measurement (specific approach) :
ascorbate oxidase
Ascorbic acid Dehydroascorbic acid (DHA)
DHA + o-Phenylene diamine → fluorescent complex → measurement at 340nm on
automated analyzer or fluorometer.
❖HPLC for Ascorbic acid quantitation involve :
• Precolumn derivatization to the fluorescent quinoxaline
• electrochemical method or
• colorimetric method
➢HPLC quantitation is a more specific approach but more time consuming .
Preanalytical treatment (addition of Dithiothreitol or Homocysteine to plasma
sample and mobile phase)of plasma sample soon after phlebotomy is needed to
prevent oxidation of Ascorbic acid by dissolved oxygen at neutral pH .
➢HPLC or gas chromatography-mass spectrometry allow measurement of Ascorbic
acid ,Total vitamin C (Ascorbic acid +Dehydroascorbic acid) and by difference DHA
together.

Biochemical functions of vitamin C in human body
1. Reversible oxidation-reduction 9. Folic acid metabolism 17. Immunological functions
(immune competence)
2. Hydroxylation of Proline and Lysine 10. Amidated peptides formation
(increased hormonal regulation)
18. Prevention of quick aging
and Cataract
3. Collagen formation 11. Conversion of cholesterol to bile
salts
19. Preventive action of chronic
diseases(Anti–diabetic)
4. Bone formation 12. Biosynthesis of Corticosteroid
hormones and steroid hormones
20. Preventive action of chronic
diseases(Anti-cancer/anti-
proliferative)
5.Tyrosinemetabolism→ Neurotransmitters
(e.g..Dopamine)andCatecholamines
(e.g.Epinephrine,Nor-epinephrine)
13 Carnitine biosynthesis(increased
metabolic energy by α and β-fatty
acid oxidation)
21. Methylation of DNA and
Histone(Decreased epigenic
regulation)
6. Tryptophan metabolism
(Serotonin formation → Melatonin)
14. . Anti-oxidant property
(response to stressful conditions)
22. Hypoxia-inducible factor
(decreased gene transcription)
7. Iron metabolism 15.Sparing action of other vitamins
(vitamin A and E)
23.Histamine degradation
8. Hemoglobin metabolism 16.Phagocytosis(promotesWBCsynthesis
/anti-microbial/anti-sepsis)
24. Anti-radiationand protection
againstozonepollution

Function of Ascorbic acid in Reversible cellular oxidation-reduction reactions
• Vitamin C can change between Ascorbic acid and Dehydroascorbic acid. It may
be involved in cellular oxidation-reduction reactions (hydrogen transport
agent).
• Most of the physiological properties of the vitamin C could be explained by
this redox-system.
• Ascorbic acid acts as a cofactor for number of mixed function oxidases in
processes in which it promotes enzyme activity by maintaining metal ions in
their reduced form(particularly iron and copper).

Function of Ascorbic acid in hydroxylation reactions
❖Ascorbic acid facilitates hydroxylation of :
• Proline and Lysine residues in collagen
• Phenylalanine to Tyrosine
• p-hydroxy phenyl pyruvic acid to Homogentisate
• Tryptamine to 5-hydroxytryptamine.
• Electron Transport in system of mammalian microsomes.
➢The Vitamin C redox system, comprises Ascorbic acid ,Dehydroascorbic acid
and the free radical intermediate monodehydroascorbic acid(the product of
one electron oxidation of Ascorbic acid).

AscorbicaciddependenthydroxylationofProlinetoHydroxyprolineandLysinetoHydroxylysine
Proline Hydroxyproline
Lysine Hydroxylysine
In Scurvy ,failure of conversion of procollagen to collagen (due to failure of hydroxylation) may
lead to a rapid destruction of the collagen intermediates.
Procollagen Proline hydroxylase
vitamin C , Fe2+ , O2
Procollagen Lysine hydroxylase
vitamin C , Fe2+ , O2

Ascorbic acid dependent hydroxylation of Proline to Hydroxyproline
COHN CO
H H
CO-HN CO
H OH
-
vvvvvvvvv
-
vvvvvvvvv vvvvvvvvv-
vvvvvvvvv-
Protein
Proline
Hydroxyproline
CO2
α-Ketoglutarate O2
Succinate H2O
Prolyl hydroxylase Ascorbic acid

Function of Ascorbic acid in cross linking Proline to Hydroxyproline and
Lysine to Hydroxylysine in procollagen
The pro-α chains are processed by a number of enzymatic steps within the lumen of RER while
the polypeptides are still being synthesized. Proline and Lysine residues found in the Y-position of
the-Gly-X-Y sequence can be hydroxylated to form hydroxyproline and hydroxylysine residues by
enzymes Prolyl hydroxylase and Lysyl hydroxylase respectively . These hydroxylation reactions
require molecular oxygen, Fe 2+ and reducing agent Ascorbic acid(vitamin C).

Ascorbic acid dependent hydroxylation of Lysine in procollagen

C=O
- CH2-CH2-CH2-CH2-C-H
NH
I
I
vvvv
vvvv
O=C
H-C-CH2-CH2-CH2-CH2-NH2
NH
I
I
C=O
HC -CH2-CH2-CH2-C-H
O NH
I
II
Lysine residue
I
I
I
I
Lysine residue Allysine residue
Formation of cross-links in collagen
I I
vvvv
vvvv
vvvv
vvvv
vvvv
vvvv
vvvv
vvvv
Lysine oxidase Cu 2+
O2
NH3 +H2O
Collagen chain
Collagen chain
O=C
H-C-CH2-CH2-CH2-CH2-NH
HN
Cross-link formation : the fibrillar array
of collagen molecules serves as a
substrate for Lysyl oxidase . This Cu 2+
containing extra-cellular enzyme
oxidatively deaminates some of Lysyl
and hydroxylysyl residues in collagen .
The reactive aldehydes that result
(Allysine and hydroxylallysine) can
condense with Lysyl or hydroxylysine
residues in neighboring collagen
molecules to form covalent cross-links,
thus mature collagen fibers.
C=O
NH2-CH2-CH2-CH2-CH2-C-H
NH

Tissues dependent on Ascorbic acid for their functional activities
Ascorbic acid is required for the functional activities of fibroblasts and consequently for the
formation of mucopolysaccharides (MPS)of connective tissues , osteoid tissues, dentine and
intracellular cement substances of capillaries.

Role of Ascorbic acid in formation of bone
and intracellular substances
in the bone ,teeth and cartilage

Functions of vitamin C in collagen formation in the human body
❖Post-translational modification of Procollagen to collagen:
• Ascorbic acid functions as a coenzyme in hydroxylation of :
1. Proline to hydroxyproline catalyzed by prolyl hydroxylase .
2. Lysine to hydroxy lysine catalyzed by lysyl hydroxylase .
• Cross linking hydroxyproline to hydroxy lysine is essential for tensile strength
of connective tissue/ fibers.
• These reaction is dependent on vitamin C ,molecular oxygen and α-
ketoglutarate.
➢Vitamin C is necessary for maintenance of normal connective tissue such as
osteoid, collagen and intracellular cement substance of capillaries thus helps
in wound healing process.

Procollagentriplehelixformation:Assemblyof3pro-alphachains
Selected Proline and Lysine residues are
hydroxylated.
Selected hydroxy lysine residues are
glycosylated with Glucose and Galactose .
Three pro-α chains assemble.
Interchain and intrachain disulphide bonds
form at the C-terminal pro-peptide extension.
A triple-helix is formed and procollagen is
produced.
The procollagen molecule is secreted from a
Golgi vacuole into the extra cellular matrix.
Steps in synthesis of Procollagen triple-helix formation
Procollagen
Proline
hydroxylase and
Lysine
hydroxylase
require molecular
oxygen, Fe 2+ and
Ascorbic acid.

Steps of Collagen formation in bone and cartilage
Genes for pro-α1 and pro-α2 chains are transcribed into m-RNAs .
Selected Proline and Lysine residues are hydroxylated.
Selected hydroxylysine residues are glycosylated with Glucose and Galactose .
Three pro-α chains assemble.
Interchain and intrachain disulphide bonds form at the C-terminal pro-peptide extension.
A triple-helix is formed and procollagen is produced.
The procollagen molecule is secreted from a Golgi vacuole into the extra cellular matrix.
TheN-terminalandC-terminalpro-peptidesarecleavedbyprocollagenpeptidase,producingtropocollagen.
Selfassemblyoftropocollagenmoleculesintofibrils,withsubsequentcross-linkingtoformmaturecollagenfibers.

Collagen formation in bone and cartilage
In the case of Ascorbic acid deficiency , hydroxylation by Prolyl and Lysyl hydroxylase is impaired.
Formation of Interchain H-bond and stable triple-helix is impaired. Collagen fibers cannot be
cross-linked greatly decreasing tensile strength of assembled fiber. Vitamin C is therefore required
for the maintenance of normal connective tissue as well as for wound healing.

Mechanism of collagen formation
• Bone matrix: consists organic matrix containing collagen ,inorganic calcium,
phosphate etc.

Deficiency of vitamin C(scurvy) and collagen formation
Vitamin C deficiency(scurvy)
no cross linking (lack of conversion of Proline to hydroxy proline and Lysine to
hydroxy lysine) in procollagen
Defective collagen formation
Connective tissue weak/fragile(decrease in tensile strength of assembled fiber)
Subcutaneous extravasation of blood due to capillary fragility
Bruises on the limbs.

Defective Bone formation in scurvy
Scurvy(dietary deficiency of
vitamin C)
Failure of the osteoblasts to
form intracellular substance,
osteoid.
The deposition of bone is
arrested due to unavailability
of normal ground substance.
Scorbutic bone is weak and
fractures easily. Hemorrhage into joint cavities. Painful joints
may prevent locomotion of the patients.

Bone density and bone quality
❖Vitamin C and vitamin B12:
• are essential nutrients to maintain bone density and bone quality.
• affect bone quality and determine collagen cross-link formation(bone quality
determinant).
• Deficiency /insufficiency of vitamin B12 induce mild elevation in levels of
plasma Homocysteine and deteriorates normal collagen cross-link formation
by enzyme Methylene tetrahydrofolate reductase(MTHFR → vitamin C
dependent enzyme) .
❖Dietary sources of vitamin C in osteoporosis :
• Amla (700mg/100g)
• Guava (300mg/100gm)

Clinical manifestations of vitamin C deficiency(scurvy) related to
defective collagen formation
❖Vitamin C deficiency →no cross linking (lack of conversion of Proline to
hydroxy proline and Lysine to hydroxy lysine) in procollagen → collagen
formation defective →connective tissue weak/fragile.
❖Clinical manifestations of vitamin C deficiency :
• Osteoporosis (osteoid activity needs collagen and mucopolysaccharides)
• Hemorrhage
• Wound healing delayed
• Weak dentine
• Bleeding gums
Scurvy: Bleeding gums and poor
dentine in an adult
Weak and bleeding dentine in
pediatric scurvy patient

Functions of Vitamin C in Tyrosine degradation
Transamination ,PLP
Tyrosine p-hydroxyphenylpyruvate
Homogentisic acid
➢Vitamin C(cofactor) helps in oxidation of para-hydroxy phenyl pyruvate
to Homogentisic acid by P-hydroxyphenylpyruvate hydroxylase(4-hydroxy
phenylpyruvate dioxygenase).
p-hydroxyphenylpyruvate hydroxylase
O2
CO2
α- Ketoglutaric acid Glutamic acid
Ascorbic acid
Cu2+

Function of Ascorbic acid in biosynthesis of adrenal hormones via Dopamine β-hydroxylase
Tyrosine
O 2
H4-Biopterin Tyrosine hydroxylase(Cu 2+ containing enzyme)
H2-Biopterin
H2O
Dihydroxyphenylalanine(DOPA)
PLP DOPA decarboxylase
CO2
Dopamine
O2 Dopamine-β-oxidase/ β-hydroxylase
Ascorbate
Dehydroascorbate
H2O
Norepinephrine
S-Adenosyl Methionine (~CH3) Phenyl ethanolamine N-methyltransferase
S-Adenosyl Homocysteine
Epinephrine
*Catecholamines and
neurotransmitters
*
*
*
Methyl group donor
←Rate limiting step
←Entry of Dopamine into chromaffin granule pheochromocytes
Synthesis of Catecholamines in adrenal medulla pheochromocytes
and neuroglial cells of sympathetic neurons
Cu 2+
SAM→
Fe2+, NADPH
Mitochondria
Cytosol
Vesicles
/granules
Cytosol

Functions of Vitamin C in Tyrosine Metabolism
Tyrosine Metabolism
L-Tyrosine
3,4 –Dihydroxyphenylalanine
(DOPA)
Dopamine
Norepinephrine
Tyrosine catabolism
p-Hydroxyphenyl pyruvate
Homogentisic acid
Maleylacetoacetate
Dopamine oxidase/
mono-oxygenase
Cu 2+
Ascorbate
In Tyrosine metabolism, DOPA is
decarboxylated in a reaction requiring
pyridoxal phosphate to form Dopamine, which
is then hydroxylated by Dopamine beta-
hydroxylase (Dopamine mono-oxygenase)to
Norepinephrine in a reaction that requires
Ascorbate and copper (Cu 2+) as a cofactor.
Tyrosine is transaminated to p-Hydroxy phenyl
pyruvate. p-Hydroxy phenyl pyruvate is then
oxidized to Homogentisic acid by p- hydroxy
phenyl pyruvate hydrolase(dioxygenase) with
Ascorbate, Cu 2+. Homogentisic acid is oxidized
to Maleylacetoacetate by Homogentisic oxidase
(dioxygenase)which requires Ascorbate , Fe2+ .
Ascorbate
Fe 2+
p-Hydroxyphenyl pyruvate
Hydroxylase/dioxygenase
Homogentisate
oxidase/dioxygenase
Cu 2+
Ascorbate
Fumarate + Acetoacetate
Tyrosine transaminase
Fumaryl acetoacetate
L-Tyrosine
↑↓Maleylacetoacetate isomerase
Fumaryl acetoacetate
hydrolase
PLP
O2
CO2
O2
Glutathione
Tyrosine hydroxylase
PLP
CO2
H4-biopterine
H2-biopterine
O2
DOPA-decarboxylase
Epinephrine
SAM

Functions of Epinephrine
Glucogeniceffectof
Epinephrine(increasesblood
sugarby):
Stimulationofglycogenolysisin
theliverandmuscle
Increasinggluconeogenesis in
liver
Decreasingperipheralutilization
ofglucose
Inhibitinginsulinreleasefrom
thepancreas
Releasingfattyacidsfrom
adiposetissueintocirculation
andchannelingfattyacidsto
hepaticgluconeogenesis
Effectsofepinephrineon
visceralsmooth muscles
(epinephrinecausesrelaxation
ofsmoothmusclesof):
Bronchioles(usedasa
bronchodilatorintreatmentof
asthma)
Urinarybladder
Gastrointestinaltract
Epinephrinecausescontraction
ofthesphinctersofstomachand
bladder.
Effects of Epinephrine on
other hormones
Acting via β-adrenergic
receptors , Epinephrine
increases the secretion of
Glucagon ,Insulin,
Thyroxine, Calcitonin
Parathyroid hormone,
Renin and Gastrin.
Inhibits insulin secretion
from pancreas by acting via
α-adrenergic receptors.
InseveredeficiencyofvitaminC,
allabovefunctionsofEpinephrine
maybeaffected.

Functions of Tyrosine ,Epinephrine and Norepinephrine
Functions of Tyrosine Functions of epinephrine Functions of norepinephrine
Synthesis of Proteins Raises the systolic blood
pressure but lowers diastolic,
Mean arterial pressure not
altered , pulse increased
Raises both systolic and
diastolic blood pressure
Synthesis of Melanin Increases the heart rate Slows the heart rate
Synthesis of Thyroid
hormones (T3 ,T4)
Increases cardiac output
(stimulates cardiac muscle
contraction)
Slightly reduces cardiac output
Synthesis of Dopamine Reduces the total peripheral
arterial resistance(generalized
vasodilator)
Increases the total peripheral
arterial resistance,
(generalized vasoconstrictor)
Synthesis of Epinephrine
and Norepinephrine
Neuro transmitter in the brain
and autonomous nervous
system
Neuro transmitter in the brain
and autonomous nervous
system
➢ InsevereScurvy,synthesisconsequently functionsofEpinephrineandNorepinephrinemaybeaffected.

Functions of Ascorbic acid in biosynthesis of Neurotransmitters
Rate limiting step →
Synthesis of Catecholamines in adrenal medulla and
sympathetic neurons
←Entry of Dopamine into
chromaffin granules.
Dopamine , Norepinephrine and Epinephrine are neurotransmitters(amine class) and derived from Tyrosine.
Serotonin (5-hydroxy tryptamine-5-HTP ) is also a neurotransmitter(amine class) but derived from Tryptophan.
Biosynthesis of these neurotransmitters is facilitated by Ascorbic acid. (one of sites of biosynthesis →CNS)

Functions of Vitamin C in Tryptophan Metabolism
Tryptophan + vitamin C →5-Hydroxy Tryptophan (5-HTP)→→ Serotonin formation
➢Ascorbic acid is necessary for the hydroxylation of Tryptophan to 5-hydroxy tryptophan .
This reaction is required for the formation of Serotonin.
➢Serotonin is converted to melatonin in the pineal gland via acetylation and methylation.
❖Functions of Serotonin:
1. Excitory activity/stimulator of cerebral activity
2. Excitory nerve impulse
3. Pain perception
4. Regulation of sleep ,appetite ,temperature ,blood pressure ,cognitive functions and mood
5. Motility of gastro-intestinal tract
6. Contraction of bronchioles and smooth muscles
7. Vasoconstriction
✓InsevereScurvy,synthesisconsequently functionsofSerotoninmaybeaffected.
Tryptophan hydroxylase
↓

Deficiency of vitamin C affects synthesis and functions of Serotonin
❖Functions of Serotonin:
1. Excitory activity
2. Excitory nerve impulse
3. Pain perception
4. Regulationofsleep,appetite,
temperature,bloodpressure,cognitive
functionsand,mood
5. Motility of gastro-intestinal tract
6. Contraction of bronchioles and
smooth muscles
7. Vasoconstriction
Inseverescurvy,formationand
functionsofSerotoninareaffected.
Deficiency of Serotonin
produces depressant effect.

Function of Vitamin C in synthesis of Serotonin in Tryptophan Metabolism
Serotonin is synthesized from Tryptophan. Tryptophan is hydroxylated by Tryptophan
hydroxylase (O 2, 5-METHF , Ascorbate , B12, Fe 2+requiring reaction) to form
5-hydroxytryptophan (5-HTP) . The product 5-HTP is decarboxylated to Serotonin, which is also
degraded by MAO. Acetylation of Serotonin followed by methylation in the pineal gland forms
a hormone Melatonin .
Argentaffin cells/
Serotonin producing
cells/ Kultchitsky’s
cells of Intestinal
mucosa, stomach,
central nervous
system and platelets
Tryptophan
5-Hydroxytryptophan
Serotonin
Tyrosine hydroxylase
5-hydroxytryptophan
decarboxylase
O 2
H2O
PLP
CO 2
Zn 2+
Ascorbate, Vit B12,Fe 2+
Melatonin
H 4 –biopterin
H2 -biopterin
N-acetyl serotonin
Serotonin-N-acetylase
N-acetyl serotonin-o-methyl
transferase
SAM
S-Adenosylhomocysteine
Acetyl CoA
CoASH
Liver Phenylalanine hydroxylase can
catalyze hydroxylation of Tryptophan.

Biosynthesis of Melatonin
L-Tryptophan 5-Hydroxytryptophan Serotonin
N-Acetyl-Serotonin
Melatonin(N-acetyl methoxy serotonin)
O 2
Hydroxylase
NADPH
CO 2
5-Hydroxytryptophan
decarboxylase
5-Hydroxy
tryptamine
(5-HT)
Serotonin N-acetylase
Acetyl –CoA
CoA-SH
S-Adenosyl methionine
S-Adenosyl Homocysteine
~CH3 N-acetyl serotonin-o-methyl
transferase
Functions of Melatonin : participates in diurnal biological rhythm and mediates in the effect of light
on seasonal reproductive cycles. Synthesis and hence functions of Serotonin and Melatonin are
Vitamin C dependent. Hence in severe scurvy, synthesis consequently functions of Serotonin and
Melatonin are affected.
Vit C
Synthesis and secretion of
Melatonin by pineal gland
and is regulated by light.
Pyridoxal
phosphate
5-hydroxyindol
aceticacid(5HIAA)
MAO

Role of Ascorbic acid in Iron absorption by facilitating reduction
of ferric iron(Fe3+) to Ferrous iron (Fe2+)of oxidases

Ascorbic acid acts as a cofactor for a number of mixed function oxidases in
processes in which it promotes enzyme activity by maintaining metal ions in
their reduced form(particularly iron and copper).

Vitamin C in facilitates dietary iron metabolism
• VitaminC(beingreducingagent),keepsironin (Fe2+ )ferrousstate.
• VitaminC,facilitatesreductiondietaryinorganicferriciron(Fe3+)toferrousstate(Fe2+ )instomach,whichispreferentially
absorbedfromthegut.IronisalsoabsorbedbyformingwatersolubleFe-ascorbatechelate.VitaminCenhancesiron
absorptionfromtheintestineinnormaloriron-deficientpatientsby10%byadministrationofvitaminC.
• Onlyferrousandnotferricformofironisabsorbed.Ferricironisreducedtoferrousironbyferricreductase,anenzyme
presentonthesurfaceofenterocytes.Ferrousironintheintestinallumenbindstomucosalcellprotein,calleddivalent
metaltransporter-1(DMT-1).Thisboundironisthentransportedintothemucosalcells.Therestunabsorbedironis
excreted.
• VitaminChelpsinformationoftissueferritin(storageformofiron).Insidemucosalcells,ferrousironisoxidizedtoferric
statebyenzymeFerroxidaseandiscomplexedwithapoferritintoformferritinwhichiskeptinmucosalcellstemporarily.It
alsohelpsinmobilizationofironfromstorageformferritin.
• VitaminCreducestheferricironofplasmaproteinforminganactivatedcomplex whichthentakespartinelectron
transport (mitochondria)ferrousironform.IntheinitialstageofETC, ATP,NAD+and NADP+stimulatetheprocess.
➢Iffreetransferrinispresentinplasma,ironcrossescellmembraneinferrousform.Inblood,itisre-oxidizedtoferricstateby
ceruloplasminorferroxidaseIIand Ferricironisthentransportedbytransferrin.
➢Iftransferrinissaturatedwithiron,anyironaccumulatedinmucosalcellislostwhenthecellisdesquamated.
➢Itassists inthereconversionofmet-haemoglobintohaemoglobinand degradationdietaryHemetobilepigments.

IronfromNon-haem
inorganiccompounds
fromfood
Gastric HCl
organic acids
Fe3+ (free iron)
Ascorbic acid
Cysteine
Glutathione
Fe2+
Apoferritin
Ferritin
(Fe3+ )
Fe3+
Ferro-oxidase Fe 2+
Fe2+
Apo transferrin
Transferrin
Fe3+
Ceruloplasmin
(ferroxidase II)
Fe2+
Lumen of Gastro-
intestinal tract
(GIT)
Mucosal cells of
Gastro- intestinal
tract(GIT)
Plasma Tissue
Storage in
Liver:
Ferritin(Fe3+ )
Hemosiderin
Utilization :
Bone marrow (Hb)
Muscles (Mb)
Other tissues:
Cytochromes
Non-heme iron
Ferro
reductase
(Fe3+ )
↕
Haem
iron
from
food
Bile pigments
Absorption ,Transport ,storage and utilization of food iron
Iron is one way
compound.

Role of vitamin C in dietary iron absorption
Entryofironinnon-hemeferricionform(boundtoprotein/organicacid)orhemeforminstomach.
Release of ferric ions(Fe3+ ) from non-heme proteins by gastric HCl and organic acids.
Reduction of ferric iron to ferrous iron(Fe2+ ) of dietary non-heme proteins by Ascorbic acid,
Cysteine and reduced Glutathione.
Absorption of ferrous form(soluble) of iron in stomach. Absorption of iron also by forming
water soluble Fe-ascorbate chelate..
BindingofFerrousironintheintestinallumen tomucosalcellprotein,calleddivalentmetaltransporter-
1(DMT-1)→Transportof boundirontothemucosalcells→Excretionoftherestunabsorbediron.
Dietary Heme-irons are absorbed directly and degraded as bile pigments .
Ascorbic acid assists degradation haemoglobin to bile pigments.

Mechanism of dietary iron absorption facilitated by vitamin C
Dietaryiron,whichismostlyinferricform(Fe3+ )isreducedtoferrousstate(Fe2+ )instomachbygastricHCland
dietaryreductantsuchasAscorbicacidandCysteine.VitaminCbeingareducingagent,itfacilitatesthe
absorptionofdietaryironbyreducingferricirontoferrousform.Ferrousformissolubleandreadilyabsorbed.
Thus,vitaminCenhancesironabsorptioninstomachandduodenumbykeepingironintheferrousform.

Mechanism iron absorption facilitated by Ascorbic acid redox-system
Ascorbic acid facilitates the absorption of non-heme iron from intestine by reducing iron to its
ferrous state . Lack of vitamin C causes disturbance in iron absorption and its functions leading
to development of hypochromic and microcytic anemia in scurvy . Absorption of Fe both in
normal or Fe -deficient patients increased by 10% after administration of vitamin C .
The Vitamin C redox system, comprises ascorbic acid ,dehydroascorbic acid and the free radical
intermediate monodehydroascorbic acid(the product of one electron oxidation of ascorbic acid).

Functions of DivalentMetalTransporter-1(DMT-1)are facilitated
by Ascorbic acid redox-system :1
❖In the mucosal cytoplasm , there is carrier called DivalentMetalTransporter-
1(DMT-1) whichisalsocalled intra-cellular iron carrier (IIC).
❖DivalentMetalTransporter-1(DMT-1)/Intracellular iron carrier (IIC) delivers
certain amount of iron to *:
1. mitochondria.
2. Apoferritin which binds to Fe3+ to form ferritin.
3. Apo-transferrin (a plasma β-globulin across the serosal cell membrane).
❖Intracellular iron carrier (IIC) holds Fe2+ in either protein bound or chelated
forms which represents the carrier iron pool in the intestinal cell.
❖Presence of sufficient amount of Fe in carrier iron pool, keeps the IIC nearly
or totally saturated and consequently reduces further Fe absorption
(Mucosal block theory).

FunctionsofDivalentMetalTransporter-1(DMT-1)arefacilitatedbyAscorbicacidredox-system:2
Ironinferritinisreleased,thencrossesmucosalcellwiththehelpofFerroportin(FP→transportprotein).This
canhappenonlywhenthereisfreetransferrininplasmatobindtheiron.Ferroportin(negativelyregulatedby
thehormoneHepcidin√releasedfromliverwhenbodyironlevelsarehigh)–majorcontrolonironabsorption.
Ascorbic acid redox-system(*) facilitate functions of Divalent Metal Transporter -1(DMT-1),
Ferroportin (FP) , Ferritin, Transferrin ,Ceruloplasmin ,Heme carrier protein (HCP1) and microsomal
heme oxygenase in iron metabolism.
*
*
*
*
Function of
Heme carrier
protein (HCP1):
help in intestinal
uptake of heme
Feisreleased
byactionof
microsomal
heme
oxygenase.
1
2
3
√

Function of Hepcidin in iron metabolism in duodenal enterocytes
Hepcidin:
1. Synthesized by liver cells and involved in killing of bacteria.
2. Coded by HAMP gene on chromosome 19.
3. Decreases surface expression of Ferroportin (responsible for moving iron across cell
membranes and its function is facilitated by ascorbic acid ).
4. Production increased by high iron stores and also by inflammation .

Role of Divalent Metal Transporter -1(DMT-1) in dietary iron absorption is facilitated by vitamin C
Only ferrous and not ferric form of iron is absorbed . Ferric iron is reduced to ferrous iron by
ferric reductase (an enzyme present on the surface of enterocytes) using Vitamin C as a
cofactor. Ferrous iron in the intestinal lumen binds to mucosal cell protein ,called Divalent
Metal Transporter -1(DMT-1).This bound iron is then transported into the mucosal cells . The
rest unabsorbed iron is excreted. Inside mucosal cells ,iron is oxidized to ferric state ,and is
complexed with apoferritin to form ferritin which is kept in mucosal cells temporarily.

Role of vitamin C in iron metabolism in mucosal cells
Thus, Ascorbic acid helps in formation of tissue ferritin (storage form of iron) and
mobilization of iron from ferritin.
Entry of absorbed ferrous iron ( Fe 2+) in the mucosal cells.
Oxidation ferrous iron (Fe 2+) to ferric form (Fe 3+) by enzyme ferroxidase I and II (copper
containing enzymes)using Dehydroascorbic acid (DHA).
Formation of Ferritin by binding of iron in its ferric state ( Fe 3+) to apoferritin inside mucosal
cells (Ferritin is temporarily storage form of iron in the mucosal cells and Ascorbic acid redox-
system maintains its iron of in oxidized status).
Reduction of Ferriciron(Fe3+) to ferrousiron(Fe2+) of Ferritin byferricreductase(anenzymepresenton
thesurfaceofenterocytes)usingVitaminC asacofactor.
Crossing of iron in ferrous form (Fe2+) from mucosal cell membrane to plasma.

Function of vitamin C in iron metabolism in mucosal cells
The iron ( Fe 2+) entering the mucosal cells by absorption is oxidized to ferric form by enzyme
ferric oxidase I and II(copper containing enzymes) using oxidized vitamin C(DHA). Inside
mucosal cells ,iron in its ferric state is complexed with apoferritin to form ferritin which is
kept in mucosal cells temporarily. Thus, Ascorbic acid is necessary for the formation of tissue
ferritin . Ascorbic acid also helps in mobilization of iron from its temporary storage form
Ferritin. Ferricironisreducedtoferrousironbyferricreductase(anenzymepresentonthesurfaceof
enterocytes)usingVitaminCasacofactor.

Transportofironinplasmafacilitatedbyascorbicacidredox-system
Reduction of ferric iron (Fe3+) of ferritin to ferrous(Fe2+) by enzyme ferro-reductase using
Ascorbic acid redox-system. Transport of iron across the basolateral membrane of intestinal
enterocytes into circulation by Ferroportin(IREG1 =iron regulated gene1→a transport protein).
Entry of the iron liberated from the ferritin of mucosal cells to the plasma in ferrous state(Fe2+).
Oxidation of ferrous(Fe2+) to ferric form (Fe3+ )by copper containing protein ceruloplasmin
(which posses ferroxidase property)or by Hephaestin (a protein cuproprotein ferroxidase II) in
plasma . Function of ceruloplasmin is assisted by Ascorbic acid redox-system.
Binding of Ferric iron (Fe3+ ) with a specific iron binding protein namely Transferrin or
Siderophilin.
Each Transferrin molecule can bind 2 atoms of ferric iron.
Concentration of Plasma Transferrin =250mg/dl
Plasma Transferrin can bind with 400mg of iron/dl of plasma. This is known as iron binding
capacity (TIBC) of plasma. In Scurvy , TIBC is elevated.

Excretion of Iron from human body and Vitamin C as an adjuvant therapy in
anaemia
Liver , spleen and bone marrow contain much ferritin. Iron is not excreted in urine but lost
from human body via bile pigments in feces , in menstrual blood and gastrointestinal bleeding
(in peptic ulcer , diverticulosis or malignancy). Rate of loss of iron is doubled or tripled during
menstruation . Therefore ,dietary iron requirement menstruating women is higher than men.
Ascorbic acid should be supplemented along with iron tablets(100 mg) as an adjuvant therapy
to facilitate iron absorption . Unabsorbed iron may generate free radicals and hence it is
advisable to give anti-oxidants(vitamin C and E) to prevent free radical generation.
Mucosal block theory: when
adequate quantity of iron
stored , absorption is decreased

Vitamin C
Role of Ascorbic acid in Heme synthesis
VitaminCreducesinorganicferricironto ferrousstate,whichispreferentiallyabsorbedfromthegut.Italso
helpsinmobilizationofironfromitsstorageformferritin.Thefinalstepinhemesynthesisinvolves
incorporationofferrousironintoprotoporphyrinIX.Thisreactioniscatalyzedbymitochondrialferrochelatase
orhemesynthasewhichusesAscorbicacid(vitaminC)asacofactor. Vitamin C deficiency(Scurvy) leads
to microcytic and hypochromic anemia.

Function of vitamin C in iron and hemoglobin metabolism

Anemia in vitamin C deficiency
❖Vitamin C deficiency leads to :
• Microcytic (size of RBCs much smaller) and Hypochromic( much reduced
hemoglobin content) anemia.
• Poikilocytosis and anisocytosis.
❖The causes for anemia associated with Scurvy may be due to :
a. Loss of blood by hemorrhage.
b. decreased iron absorption.
c. Decreased tetrahydro folic acid.
d. Accumulation of met-hemoglobin.

Functions of vitamin C in folic acid metabolism
❖Vitamin C :
• regulates the
conversion of folic acid
to folinic acid (so called
citrovorum factor).
• maintains folic acid
reductase in its active
form by keeping folic
acid in the reduced
Tetrahydrofolate (FH4)
form.
• In combination with
folic acid , Ascorbic acid
helps in maturation of
red blood cells.
Vitamin C
Vitamin C

Function of vitamin C in folic acid metabolism
Folic acid + vitamin C →Tetrahydrofolate (THF)
➢Ascorbic acid helps enzyme folate reductase to reduce folic acid to tetra
hydro folic acid. Thus, it helps in maturation of RBC.
❖Tetrahydrofolate (THF) plays role in:
▪ Amino acid & protein biosynthesis
▪ Synthesis of purine → DNA synthesis →wound healing/RBC maturation
➢Deficiency of vitamin C →deficiency of folinic acid → delayed wound healing .
➢Deficiency of vitamin C →deficiency of folinic acid →no RBC maturation →
macrocytic anemia.
Folic acid reductase
↓

One carbon metabolism dependent on THF
Glycine , Serine Histidine etc.
One carbon (1C ) donors
One carbon moiety (1C)
accepted for synthesis of
One carbon (1C moiety)
Methyl, Formyl etc.
THF 1C-THF
Ascorbic acid facilitates
synthesis of THF
Amino acids
Glycine, Serine
Purine (2,8 carbons) →
DNA ,RNA
Pyrimidine nucleotide –
Deoxy-thymidylic acid (dTMP)→DNA
Choline,
Ethanolamine
N-Formyl methionine→
initiator of protein biosynthesis

Function of vitamin C in Tetrahydrofolate (THF) formation
Tetrahydrofolate (THF) plays
role in:
▪Amino acid & protein
biosynthesis
▪Synthesis of purine → DNA
biosynthesis →wound healing
and RBC maturation
➢ Deficiency of vitamin C→
deficiency of folic acid →
no RBC maturation →
Macrocytic anemia

Megaloblastic anemia
Decreased RBC (decreased hematocrit),
increased MCV , hyper segmented
Neutrophils , Increased Homocysteine

Nutritional anemias : microcytic and megaloblastic anemia
Type of Nutritional anemia Cause/s MCV(μm 3)
Normocytic Protein-energy malnutrition =80-100
Microcytic Deficiency of Ascorbic acid
Deficiency of iron
Deficiency copper
Deficiency in pyridoxine
< 80
Macrocytic Deficiency of vitamin B12
Deficiency of folate
>100
Normal mean corpuscular volume (MCV) for adults with age more than 18 years = 80-100 μm3

Function of vitamin C in peptide synthesis
• Many peptide hormones contain carboxyl terminal amide which is derived
from terminal Glycine.
Glycine of Terminal amide + Vitamin C Hydroxylation of Glycine
Peptidyl glycine hydrolase
↓

Function of vitamin C in post-translational modification of proteins
❖Vitamin C dependent post-translational modification of proteins:
▪ Proline and Lysine hydroxylation: by enzyme Prolyl hydroxylase and Lysyl
hydroxylase respectively . The most important hydroxylated proteins are
collagens.
▪ Carboxy terminal amidation : The donor of the terminal amide group is
Glycine . Several peptide hormones (Gastrin,CCK,Oxytocin,Vasopressin,
Thyrotropinand corticotropin) have C-terminal amidation .

Function of Ascorbic acid dependent Peptidyl glycine α-amidating mono-
oxygenase
Inactivationofmanypeptidehormones(Gastrin,CCK,Oxytocin,Vasopressin,Thyrotropinandcorticotropin),
hormonereleasingfactorsandneurotransmitters,enzymePeptidyl glycineα-amidatingmonooxygenase
playscriticalrole.ThisenzymerequiresAscorbate,molecularoxygenandcuprousions(Cu2+).Itcleaves
carboxy-terminalwithhelpofmolecularoxygen.Aminogroupisreattainedasaterminalamide whilerestis
releasedasGlyoxylate.

Function of vitamin C in peptide hormone synthesis
Many peptide hormones (Gastrin,CCK,Oxytocin,Vasopressin,Thyrotropinandcorticotropin) contain
carboxy-terminal amide which is derived from terminal Glycine. Hydroxylation of Glycine is
carried out by Peptidyl glycine α -amidating monooxygenase (peptidyl-α-hydroxyglycine)
which requires vitamin C.

Function of vitamin C in peptide synthesis

Function of Ascorbic acid in hydroxylation of Cholesterol during Bile acids biosynthesis
Cholesterol
NADPH + H+ 7-α-hydroxylase
O2 Ascorbic acid
NADP+
7 –Hydroxycholesterol
Cholic acid Chenodeoxycholic acid
Glycine Taurine Glycine or Taurine
Glycocholic acid Taurocholic acid
intestinal bacteria
Deoxy cholic acid Glycocheno deoxycholic acid or Tauro deoxycholic acid
BiosynthesisofBileacids:Cholesterolishydroxylatedto7–Hydroxycholesterolby7-α-hydroxylasewhichusesAscorbicacid
asacoenzyme.Thisreactionrequiresmolecularoxygen,NADPHandCytochromeP-450.It isaratelimitingstepand
synthesisofbileacidsisregulated.Scurvyleadsaccumulationofcholesterolandatherosclerosisinscorbuticanimals.
intestinal bacteria
Lithocholic acid
↙Several steps
**
*
* *
**
CytochromeP-450
* Primary bile acids
* * Secondary bile acids
Liver : site of bile
acids biosynthesis
←Rate limiting step

Function of vitamin C in cholesterol metabolism

Role of vitamin C in prevention of gall stone formation
Cholesterol +Vitamin C → hydroxylation →
7-Hydroxycholesterol →→→ Bile acids → Bile salts
➢ In Scurvy, biosynthesis and consequently functions of
bile salts are affected→ gall stone formation.
Functions of bile salts:
▪ absorption of carotene ,lipids and fat soluble
vitamins by lowering surface tension.
▪ formation of micelle with fatty acids , mono/di/
tri-acyl glycerol.
▪ choleretic action.
▪ Accelerate action of pancreatic lipase.
▪ Keep cholesterol in solution in gall bladder bile
and hence prevent gall stone formation.

Ascorbicacidfacilitatessynthesisbilesaltsandhencemaintainstheirfunctions
Accelerate
action of
pancreatic
lipase
Formation of
micelle with
fatty acids ,
mono/di/ tri
acyl glycerol
Bile salts provide only significant means for cholesterol excretion, both as a metabolic product
of cholesterol and as a solubilizer of cholesterol in bile.
Enhanced
amphipathic nature
Emulsification of fat

Biosynthesis of Corticosteroids and Steroid hormones facilitated by Ascorbic acid
Cholesterol (27C)
NADPH Desmolase(CYP11A ,p450 soc )
Ascorbic acid
Pregnenolone (21C)
3-β-Hydroxy steroid dehydrogenase
Progesterone (21C)
17-α-Hydroxylase ( CYP17)
17-Hydroxy progesterone (21C)
11-Deoxycorticosterone (21C) 11-Deoxycortisol (21C) Androstenedione (19C)
Corticosterone
Testosterone (an androgen)
Aldosterone(21C) Cortisol(21C) Estradiol ( 18C)
(a mineralocorticoid) (a Glucocorticoid) (an oestrogen)
21-α Hydroxylase
11-β-Hydroxylase( CYP11B1)
11β-Hydroxylase
(CYP11B1)
Aromatase(CYP19)
17β-hydroxysteroid
dehydrogenase
Steroid hormone synthesis
in Adrenal cortex
Peripheral
tissue
Not in
Adrenal
cortex
Ascorbic acid
has some role in
adrenal
steroidogenesis.
It is involved in
the
hydroxylation
reactions of
steroids.

Function of vitamin C in corticosteroid hormonal regulation
❖Tissue dependent on vitamin C includes:
Adrenal gland
Pituitary gland
Corpus luteum
Retina
❖High concentration of vitamin C in target tissues → Increased hormone
activity e.g.
Stressful conditions →concentration of vitamin C increases→
Hydroxylation increases →synthesis corticosteroids increases → releasing
Glucose →fight against stressful conditions.

Role of vitamin C in corticosteroid hormonal regulation
Stressful conditions
concentration of vitamin C increases in target tissue(Adrenal gland, Pituitary gland and
Corpus luteum )
Hydroxylation increases
Biosynthesis corticosteroids increases
releasing glucose
fight against stressful conditions.

Role of vitamin C in Steroid hormone biosynthesis
➢Large quantities of vitamin C are present in adrenal cortex.
➢Ascorbic acid is depleted by ACTH stimulation.
➢Ascorbic has some role in adrenal steroidogenesis . It is involved in the
hydroxylation reactions of steroids.

Steroid hormone biosynthesis in adrenal cortex
Ascorbic acid has some role in adrenal steroidogenesis . It is involved in the hydroxylation
reactions of steroids.

Role of vitamin C in Carnitine biosynthesis:1
Two reactions of Carnitine biosynthesis where Ascorbate serves as a cofactor :
1. 6-N-trimethyl-L-lysine hydrolase (6-N-trimethyl-L-lysine-3- hydroxylase
requiring alpha ketoglutarate ,Fe 2+) which hydrolyses trimethyl lysine to
3-hydroxy trimethyl lysine.
2. γ-butyro betaine hydrolase(4-butyrobetaine hydroxylase –a dioxygenase
requiring alpha ketoglutarate ,Fe 2+) which hydrolyses γ-butyrobetaine to
carnitine.
Role of Vitamin C in alpha(α)-oxidation of fatty acids : Ascorbate helps in the
action of the enzyme α-hydroxylase (a mono-oxygenase)which catalyzes the
alpha-oxidation of long chain fatty acids to form alpha-hydroxy fatty acids.

Role of vitamin C in Carnitine biosynthesis:2
FormationofCarnitineinliverbyhydroxylationofDeoxycarnitinebyDeoxycarnitine
hydroxylase(dioxygenase)withhelpofmolecularO2 vitaminC,AlphaKetoglutarate, Fe2+ .

Functions of Carnitine
Carnitine shuttle : during beta(β) and alpha(α) oxidation ,the fatty acid must be transported
across the inner mitochondrial membrane that is impermeable to CoA . Therefore , a
specialized carrier transports the long-chain acyl group from the cytosol into the
mitochondrial matrix . This carrier is Carnitine and this rate-limiting transport process is called
the carnitine-shuttle.
In vitamin C deficiency(Scurvy), biosynthesis and consequently functions of Carnitine are
affected. Lack of carnitine aggravates fatty liver formation in Scurvy .

Vitamin C deficiency leads to fatty liver
Scurvy (dietary deficiency of vitamin C)
Decreased biosynthesis of Tyrosine
Decreased carbohydrate regulation(hypoglycemia) →
weakness ,lethargy
Increased Glycogenolysis
Decreased Lipolysis(decreased Triacylglycerol
break down)→Fatty liver
Lack of carnitine
aggravates fatty liver
formation

Reactive oxygen species(ROS) and their characteristics
❖Reactive oxygen species(ROS):
▪ Superoxide anion(O2 )
▪ Hydroperoxyl radical (HOO )
▪ Hydroxyl radical (OH
.)
▪ Hydrogen peroxide(H2O2)
▪ Singlet oxygen( ¹O2)
▪ Lipid peroxide radical (ROO .)
▪ Nitric oxide(NO
.)
▪ Peroxyl nitrite (ONOO .)
.
. Important characteristics of ROS are :
➢ Extreme reactivity
➢ short lifespan
➢ Generation of new ROS by chain reaction
➢ Damage to various tissue.
Peroxidation of polysaturated fatty acids:
COOH COOH
(+)OH .
PUFA(R)
PUFA radical (R
.)

Examples of Reactive oxygen species(ROS)
-
H H
H
H
H
-
Superoxide anion (O2
- )
. Hydroperoxyl radical (HOO )
. Hydroperoxyl radical (OH )
.
Hydrogen peroxide(H2O2) Oxygen(O2) Hydroxyl ion (OH - )

Formation of intermediates from molecular oxygen
O2 O2 H2O2 OH H2O
Oxygen superoxide Hydrogen peroxide Hydroxyl radical water
O2 O2 H2O2 OH H2O
Oxygen superoxide Hydrogen peroxide Hydroxyl radical water
-
. .
e↘ e↘ e↘ e↘
- - - -
Actions of antioxidant enzymes
. .
-
Superoxide dismutase
Catalase
2-GSH(reduced Glutathione) G-S-S-G (oxidized Glutathione)
Glutathione peroxidase

Damages by Reactive Oxygen Species(ROS) in the Eukaryotic cell
ROS
DNA damage,
cell death ,
Mutation,
cancer Protein damage : loss of function
Mitochondrial
permeability
transition
Lipid peroxidation→ membrane
damage
Both ascorbate /ascorbyl radical
have low reduction potential
therefore serve the most effective
water soluble anti-oxidants in
biological system.

Anti-oxidant activity of Ascorbic acid
❖Anti-oxidant activity of Ascorbic acid would help to :
▪ Scavenge reactive oxygen species(ROS) and reactive nitrogen species(e.g. Nitric
oxide-NO).
▪ Regenerate small molecule antioxidants including α-Tocopherol(vitamin E)
reduced Glutathione ,urates and β-carotene from their respective radical
species.
▪ Maintain sulfhydryl compounds such as Glutathione in the reduced state.
▪ Prevent reactive oxygen species (ROS) binding to cellular macromolecules
(DNA, RNA, lipids and proteins) and therefore prevent damage to these
biomolecules .
➢Ascorbyl radical is relatively stable because of resonance stabilization of
unpaired electron.
➢Both ascorbate /ascorbyl radical have low reduction potential therefore serve
the most effective water soluble anti-oxidants in biological system.

Antioxidant activity of Vitamin C
❖Anti-oxidant activity of Ascorbic acid would help to :
1. Scavenge reactive oxygen species(ROS) and reactive nitrogen species(e.g. Nitric oxide-NO).
2. Regenerate small molecule antioxidants including α-Tocopherol(vitamin E), reduced
Glutathione ,urates and β-carotene from their respective radical species.
3. Maintain sulfhydryl compounds such as Glutathione in the reduced state.
4. Prevent ROS binding to cellular macromolecules (DNA ,RNA ,lipids and proteins)and their damage.
1
2
3

Function of Vitamin C in sparing Vitamin A and E
• ANTIOXIDANT PROPERTY
• SYNEGESTIC ACTIO ----SPARING ARCTION WITH VITAMIN A ,CAROTENE ,& E
Vitamin C is one of a group of nutrients that includes vitamin E and β-carotene ,which are known
as anti-oxidants. Consumption of diets rich in these compounds is associated with a decreased
incidence of some chronic diseases ,such as coronary heart disease and certain cancers. Clinical
trials involving supplementation of antioxidantcombinations forpreventionofcancerorcardiovascular
diseaseshaveprovedtheir beneficialeffects.

Ascorbylation of lipid peroxidation products by Vitamin C
Oxidized low density lipoproteins (LDL) formed by action of free radicals ,promote
atherosclerosis and Coronary heart disease(CHD) . Vitamin C is thought to be involved in the
prevention of atherosclerosis and coronary heart disease by preventing oxidation of Low
density lipoproteins (LDL).

Function of vitamin C in phagocytosis
Ascorbic acid stimulates phagocytic action of leucocytes and enhance the
formation of antibodies(immunoglobulins).

Immunological functions of Ascorbic acid (Immunocompetence)

Virus(COVID-19) neutralization by Ascorbic acid

The possible Beneficial effects of Ascorbic acid in
management of Covid -19 (?)

Antioxidant Function of Vitamin C in periodontal disease

Toxicity of Free radicals
Age spots ,wrinkles , sagging
Eye :Diabetic retinopathy ,cataract, age
related macular degeneration
Brain:Parkinson’sdisease,Alzheimer's
disease,amyotrophiclateralsclerosis
Chest : arrhythmia , cardiac infarction, high
blood pressure
Air tube : bronchial
asthma, inhalation injury
Abdomen : gastric ulcer, fatty liver, ischemic
colitis
Lower abdomen : kidney failure, uremia
Human body : Aging , diabetes ,
allergy, rheumatic disease , cancer,
arterial sclerosis
✓ Vitamin C and other antioxidants offer some protective effect against toxicity of free
radicals.

Functions of vitamin C as an antioxidant in prevention of diseases
❖Daily intake of Vitamin C prevents :
• anemia: by keeping iron of heme-protein in ferrous form(reduced form).
• quick aging
• cataract
• increase in cholesterol (prevents atherosclerosis / coronary heart diseases /
circulatory diseases/stroke)
• chronic diseases (such as cold to cancer)/ aniline dye induced bladder cancer
• varicose veins
• gall stone formation
• bacterial infections/inflammatory diseases
• Influenza
• allergy
Ascorbic acid spares vitamin A ,Vitamin E and some B-complex.

Function of vitamin C in prevention of quick aging
Free radicals are closely associated with the
various biochemical and morphological
changes that occur during normal aging. Daily
intake of Vitamin C reduces the risk of cataract
formation and quick aging by its antioxidant
property.
Increased exposure to oxidative stress
contributes cataract formation which is
mostly related to aging.

Prevention cataract formation by Ascorbic acid (mechanism)
Glucose
NADPH GSSG Ascorbate GSH
NADP + GSH Dehydroascorbate GSSG
Sorbitol
Free
radicals
Detoxified
products
Age , Glycation, nitric acid metabolism ,
prostaglandin metabolism , ROS
production in WBC, hyper-insulinoma
Vitamin C reduces the risk of cataract
formation by its antioxidant property
(prevents oxidation of Glutathione).
Ascorbyl
Radical
Superoxide dismutase
Glutathione
Peroxidase
GPX
Glutathione
reductase

Oxidative stress and Diabetes mellitus
Destruction of islets of pancreas due to accumulation of free radicals is one of the causes of
pathogenesis of insulin-dependent diabetes mellitus(IDDM) . Free radicals contribute towards
the long term complications of Diabetes Mellitus (e.g. vascular diseases / atherosclerosis,
Diabetic retinopathy, Diabetic neuropathy and Diabetic foot). Oxidized low density lipoproteins
(LDL) formed by action of free radicals ,promote atherosclerosis and CHD.
Vitamin C (being anti-oxidant) supplementation prevent severity of these complications.

Long-term complications of Diabetes Mellitus due to oxidative stress
Diabetic foot
Diabetic neuropathy
Vitamin C (being anti-oxidant) supplementation prevent the severity of these long-term
complications of Diabetes Mellitus.

Diabetic cataract
Diabetic cataract and Diabetic retinopathy
VitaminCisconcentratedinthelensofeye
/retina.RegularintakeofvitaminCreduces
riskofcataractformationand retinopathy.

Mechanism of anti-oxidant activity of Ascorbic acid

Antioxidant property of Ascorbic acid in prevention of cancer
Free radicals can damage DNA and cause mutagenicity and cytotoxicity .Thus ,free radicals
play role in carcinogenesis. The activation of protooncogenes to oncogenes is important step
in causation of cancer (e.g. activation of KRAS/BBAF protooncogene in colon cancer).
Consumption of Vitamin C reduces the risk of carcinogenesis by its antioxidant property
(prevents oxidation of Glutathione).

Antioxidant property of Ascorbic acid facilitating prevention of colon cancer
AntioxidantpropertyofAscorbicacidisassociatedwithpreventionofcoloncancerby
inhibitingnitrosamineformationfromnaturallyoccurringnitrates(whichareproduced
duringdigestion).

Role of vitamin C in Methylation of DNA and Histone
(Decreased epigenic regulation)
Reactive oxygen species (ROS) can induce mutations and inhibit DNA repair process ,that results
in the inactivation of certain tumor suppressor genes or activation of oncogenes leading to
cancer. Free radicals promote biochemical and molecular changes for rapid tumor growth.
Ascorbic acid efficiently scavenges free radicals and prevent / reduce the occurrence of cancer.
DNA hypermethylation
Histone methylations
Histone modifications
Up-regulated miRNAs
Epigenic inactivation
Tumor suppressor genes
DNA hypomethylation
Histone methylations
Histone modifications
Down-regulated miRNAs
Epigenic re-depression
Oncogenes or cancer promoting genes
ROS induced Carcinogenesis
Ascorbic acid

Effect of normoxia and hypoxia on gene transcription of HIF1-α induced genes
Hypoxia induced factor 1-alpha represents oxygen regulated subunit of transcription factor HIF-1 which
regulates the transcription of numerous genes involved in cellular response to hypoxia and oxidative stress .
Nitric oxide(NO) induces HIF-1α stabilization in human endothelial cells from umbilical cords (HUVECs) under
normoxia conditions. Vitamin C inhibits NO induced stabilization of HIF1-α in HUVECs ( PUBID: 20380593).

Representative HIF1-α regulatory genes
RepresentativeHIF1-αregulatorygenes
Role of Hypoxia-
inducible factor

Vitamin C regulates gene transcription of Hypoxia-inducible factor(HIF)
Vitamin C inhibits NO induced stabilization of HIF1-alpha in HUVECs ( PUBID: 20380593)

Prolyl Hydroxylase(PHDs) inhibition-aided therapy
Effect of vitamin C on activation of hypoxia inducible factor 1-α and 2-α gene in Thyroid
cancer cell lines : Results showed that in both cancer cell lines Vitamin C, induced a dose
dependent decrease of HIF 1-α protein level.

Vitamin C regulates somatic cell programming
Vitamin C promotes the generation of induced pluripotent stem cells (iPSCs) through
activity of Histone demethylating dioxygenase . In absence of vitamin C ,TET 1 promotes
somatic cell programming independent of TET.

Role of vitamin C in Histamine catabolism
Histidine
Histamine
N-Methylhistamine
N-methyl-β-imidazole acetate
Histamine
degradation in Liver
Ascorbic acid has beneficial effects when given
synergistically with Histamine H1 receptors
antagonist(diphenhydramine used in treatment of
allergic response and asymptomatic treatment for
upper respiratory disorders)→Anti-histaminereaction.
HistamineN-methyltransferase
SAM(-CH3)
Histaminase
Ascorbic acid
PLP
CO 2
Histidine decarboxylase
O 2
NH3
N-methyl- β -imidazole acetaldehyde
Aldehydeoxidase
O 2

Radioprotective effect of vitamin C as an anti-oxidant
Vitamin C
X-rays, Ionizingradiations(e.g.α,β,γ-
rays),radioactiveisotopes,protons
andneutronsgeneratefreeradicalsin
humantissueandcauseoxidative
stress(DNAdamage).Anti-oxidants
includingAscorbicacid arescavengers
ofradiationinduced freeradicals,
thereforereduce mutagenesisand
tissueinjury.

Radioprotective effect of vitamin C as anti-oxidant

Toxic effects of ground level ozone air pollution on the human body
Ascorbicacid,Glutathioneinproximalanddistalrespiratorytractadditionallyreactwithozone.Theyreduce
toxiceffectsofground-levelozoneairpollution.
❖Short term effects of ozone air pollution on the human body :
▪ Breathing difficulty in outdoor activities
▪ Shortness of breath
▪ Headache
▪ Throat and lung irritation
▪ Nausea
❖Long term effects of ozone air pollution on the human body :
▪ Decreased lung functions.
▪ Pre-mature aging of lungs(more susceptible to infections).
▪ Worsened symptoms of emphysema, asthma and other lung diseases
(including brochities).
▪ Inflammation and damage to lining of the lungs

Toxic effects of ground level ozone pollution on the human body
Ozone

ProtectionagainsttoxiceffectsofozonepollutionbyAscorbicacid
• Low Ozone content in the atmosphere : protection is by uric acid present in
the lining of the nasal cavity. Most individuals are able to protect against
small amounts of ozone in the atmosphere.
• Ascorbic acid , Glutathione in proximal and distal respiratory tract
additionally react with ozone .They reduce toxic effects of ozone pollution .
• Second line of defense against ozone : by α-Tocopherol and Glutathione.
• Ozone which escapes antioxidant screen can react with proteins,
carbohydrates and lipids to generate lipid peroxides to initiate destructive
chain reaction .
• High Ozone content in the atmosphere : 10-20% of individuals can have
respiratory symptoms.

• Intracellular reducing agents such as Ascorbate ,vitamin E and β-carotene
are able to reduce and detoxify oxygen intermediates.
• Consumption food rich in these antioxidant compounds have been correlated
with reduced risk of certain types of cancers as well as decreased frequency
of certain other chronic health problems.
• The effects of these compounds are in part ,an expression of their ability to
quench the toxic effects oxygen intermediates.
• Health promoting effects of dietary fruits and vegetables probably reflects a
complex interaction among many naturally occurring compounds , which has
not been duplicated by consumption of isolated antioxidant compounds.

Scurvy(deficiency of vitamin C)
Corkscrew hairs and perifollicular hemorrhage
on legs of the old man suffering from scurvy

Scurvy(deficiency of vitamin C)
Name Co-enzyme form Category(years of age) RDA
(mg/day)
Deficiency disease
Ascorbic acid No specific form Adult male(19-70 ) 90 Scurvy
Women(19-70 ) 75
Pregnancy and Lactation 150
Male Infants(1-3) 15
Male Children (4-8) 40
Female infants(1-3) 45
Female children(4-8) 65
Infants up to 6 months(AI) 40
Infants up to 7-12
months(AI)
50
Adult patients receiving
TPN
200
Total body pool of
Human male
(physiological)=
1.5 -2 g
Total body pool of
Human male
(Scurvy) = 300mg
Main reaction using the co-
enzyme :
Anti-oxidant property due to its
reducing action ,hydroxylation
of collagen.

Protracted deficiency of vitamin C leads to the classic disease of scurvy.

Causes of Scurvy( deficiency of Vitamin C)
❖Causes of scurvy :
▪ Gross dietary deficiency(unbalanced diets).
▪ Tinned food to greater extent without fresh fruits and vegetables.
▪ Infants receiving cow’s milk not supplemented.
▪ Infants receiving breast milk from deficient mother.
▪ Cooking food in frying pans, the combination of heat and large area of food in
contact with air irreversibly oxidizes the vitamin c → looses its biological activity .
▪ Iron overload.
▪ Oral contraceptive pills (WBC ,platelet and ascorbic acid reduced)
▪ Infectious diseases/fever.
▪ Alcohol dependent/smokers.
▪ Mentally ill patients /renal failure patients undergoing peritoneal dialysis or
hemodialysis.
▪ Oxidative stress(renal /liver diseases ,malignancies, congestive heart failure).

Symptoms of Scurvy(deficiency of Vitamin C)
❖Symptoms of scurvy:
• Fragile blood vessels
• Hemorrhage (petechiae →pin point Subcutaneous hemorrhages/ bleeding,
periosteal due to increased capillary fragility)
• Delayed wound healing, fatigue, aching muscles(due to muscle weakness)
• Sore gums(swollen ,spongy ,bleeding , painful)
• Poor dentine formation, pulp separated from dentine and finally teeth are lost.
• Osteoid formation defective(osteoporosis)
• Impaired bone formation → weak Bone →cannot withstand stress→ repeated
fractures
• Impaired erythropoiesis →Anemia(microcytic ,hypochromic)
• Decreased immunocompetence/ immune functions
• Sluggish hormonal functions of adrenal cortex and gonads
Symptoms of Scurvy are related to impairment in synthesis
of collagen /or the antioxidant property of vitamin C.

Clinical manifestations of Scurvy:1
• Gums: livid and swollen
• Cutaneous bleeding often begins on lower thighs as perifollicular hemorrhages and
large spontaneous bruises(ecchymoses) may arise almost anywhere on the body.
• Ocular hemorrhage
• Drying of salivary and lachrymal glands
• Parotid swelling
• Femoral neuropathy
• Edema on lower extremities
• Psychological disturbances
• Anemia(microcytic, hypochromic)
• Display Radiological changes characteristics of osteoporosis(Bayonet’s rib syndrome
in infants).
• Sudden death due to heart failure in scorbutic patients

Clinical manifestations of scurvy:2
Oral cavity in scurvy :Sore gums (swollen,
spongy, bleeding and painful).Pulp is
separated from dentine and finally teeth
are lost . Wound healing may be delayed.
Petechiae →
pin point Subcutaneous
hemorrhages on legs of
the old man suffering
from Scurvy
Weak and bleeding dentine in
pediatric scurvy patient
Scurvy: Bleeding gums and
poor dentine in an adult

Clinical manifestations of scurvy:3
Microcytic,hypochromicanemiainscurvy Corkscrew Hair (dry and coiled hair) and
small bleeding near hair follicle
Microcytic → size of RBCs much smaller
Hypochromic → much reduced hemoglobin content

Radiological features of Scurvy
ThelackofvitaminCcausesinabilitytoformadequateintracellularsubstanceinconnectivetissue
andisreflectedinswollen,tenderandbleeding/bruisedlociatjoints(alsoatotherareas).

Hemorrhagic tendency in Scurvy
Scurvy (ascorbic acid deficiency)
Defective formation of collagen
Intracellular cement substances become brittle.
Capillaries are fragile.
Tendency to bleed /hemorrhage under minor pressure.
Manifestation of Subcutaneous hemorrhage as petechiae(mild deficiency)and as large
spontaneous bruises(ecchymoses) or as hematoma(severe deficiency)

Tourniquet test for determination of hemorrhagic tendency in scurvy
➢Tourniquet test = fragility test= capillary resistance test
➢A sphygmanometer cuff is placed around the forearm and inflated. So that it
compresses the venous blood flow. This pressure is kept for 5 minutes.
➢Appearance of several Petechiae hemorrhages (20 or more /6.25cm2)may
seen on the forearm skin indicate Vitamin C deficiency . This is a very useful
clinical test.
Normal
Scurvy

Tourniquet test: diagnostic test for scurvy
Scurvy
Normal
Sphygmanometer

Internal hemorrhage in scurvy
➢In severe cases of scurvy ,hemorrhage may occur in the conjunctiva and
retina.
➢Internal bleeding may be seen as epistaxis ,hematuria or melanoma.
Subconjunctival bleeding
Splinter
Hemorrhage in
nails

Infantile Scurvy (Barlow’s disease)
• InfantileScurvy(Barlow’sdisease):manifestedininfantsbetween6to12monthsofage(periodinwhich
weaningfrombreastmilk).
• Infantilescurvydevelopswhenbabiesarebottle-fedonboiled/pasteurizedorcondensedmilk/
reconstituteddriedmilk withoutfreshfruitjuices(nosupplementationofascorbicacid).
• Theprescorbuticinfantsbecomeanorexicandlistlessforfewdays.
• Withthe beginningofthedisease,theinfantslieswithlegsdrawnupontheabdomen.
• Theinfantscreams/crieswhentouchedespeciallywhenitslegsandarmsaremovedorlifted.Infantsexhibit
bayonet’s-ribsyndrome.
• Extremetenderswellingmaybefeltattheendoflongbones.Thelongbonesareacutelypainfuldueto
hemorrhageunderperiosteum.
• Thesternummaysinkslightlyinward.
• Purpuraoccursintheskin.Thegumsareswollenandbleed.
• Iftreatmentisdelayed,dyspnea,apathy,weakness,cyanosis,convulsionsanddeathmayoccur.
• ThedietshouldbesupplementedwithvitaminCsources.Otherwise,deficiencyofvitaminCisseen.

Clinical manifestations of Infantile Scurvy (Barlow’s disease)
Infantilescurvy(Barlow’s disease) :Extremetenderswellingmaybefeltattheendoflongbones.Thelong
bonesareacutelypainfulduetohemorrhageunderperiosteum.Thesternummaysinkslightlyinward.
Bayonet’s-rib
syndrome
Edema of
lower
extremities

Bachelor scurvy
Bachelor or widowers scurvy : elderly bachelors and widowers who may
prepare their own foods are particularly prone to development of vitamin C
deficiency.
In scurvy (deficiency of vitamin C ) ,patients show bruises on the limbs as a result of
subcutaneous extravasation of blood due to capillary fragility.

Diagnosis of Scurvy
(1) Prompt improvement following administration of vitamin C.
(2) Estimation of Concentration of serum vitamin C.
(2)Tourniquet test
(3)Urine Ascorbic acid Saturation test :
Administer 5mg of Ascorbic acid /2.5 Kg body weight orally → 50% of
administered vitamin C in urine within 24 hrs.→ no scurvy (no deficiency of
vitamin C) .
Scurvy : 5mg Ascorbic acid /2.5 Kg body weight orally l→0 mg of vitamin C in
urine within 24 hrs.
(4)Intradermal test :
Intradermal injection of 2,4- dichlorophenol indophenol→ determination of
time required for decolorization i.e. reduction of dye.
Dye detained abnormally /long persistence of blue color sub-dermally→ sub
saturation of Ascorbic acid(deficiency of vitamin C).

Reference intervals of biochemical parameters related to scurvy
Parameter Physiological Severe Scurvy (Microcytic, Hypochromic anemia)
Total Serum Ascorbic acid
( Ascorbic acid +
Dehydroascorbic acid)
0.4-1.5mg/100ml
(23-85 μmol/L)
< 0.2 mg /100ml(severe deficiency)
(<11 μmol/L)
Ascorbic acid(Leucocytes) 20-53 μg/10 6 WBC
1.14-3.01 fmols /106 WBC
<10 μg/10 6 WBC
< 0.57 fmols /106 WBC
Urine Ascorbic acid 15-20mg/24hr 0
Buffy coat 15-25mg/100ml < 2 mg/100ml
Serum iron (adult)
Serum iron (children)
100 -250μg/dL
(20-30μmol/L)
50-120μg/dL
<100μg/dL
< 45μg/dL
RBC count 4.6 million / mcl < 4.1million / mcl
Hemoglobin (male)
Hemoglobin (female)
14-16gm/dL
13-15 gm/dL
<10gm/dL
Plasma/serumAscorbicacid:respondtochangesindietaryvitaminCconcentration(usedforassessment
recentvitaminCintake)andpoorindicatoroftissuelevels. Laboratory independently define its own reference levels.

Hemosiderin accumulation in Scurvy
❖Hemosiderinisahemoglobin-derivedgranularpigmentandaccumulatesintissuewhenthereisalocalor
systemicexcessofiron.Itisformedbypartialdeproteinizationofferritinbylysosomes.
❖Ironisnormallystoredwithincellinassociationwiththeproteinapoferritin,formingferritinmicelles.
Hemosiderinpigmentrepresentsalargeaggregatesoftheseferritinmicellesbutitismoreinsolublethan
ferritin.Therefore,Ironismoreslowlyreleased.
❖VisualizationHemosiderinbylightmicroscopy:
1. Goldenyelloworbrowncoloredpigment.
2. IroncanbeunambiguouslyidentifiedbyPrussianbluehistochemicalreaction.
❖ Accumulationofhemosiderin:
a. Physiological:insmallamountsin mononuclearphagocytesofliver,bonemarrowandspleen.
b. Pathogenic :Localexcessofiron→ hemosiderinaccumulation(e.g.hemorrhagewhenthereis
extensivebreakdownofabnormal RBC(hypochromicandmicrocyticresultingfromseverescurvy).
❖ ABruise:afterlysisofRBCatthesiteofhemorrhage,theredcelldebrisisphagocytosedbymacrophages.
ThehemoglobincontentiscatabolizedbylysosomeswithaccumulationofironinHemosiderin.
❖Hemosiderosispulmonis(alveolarHemosiderosis)

Deposition of hemosiderin in Scurvy
Scurvy
Lysis of abnormal erythrocytes (microcytic , hypochromic)
The red cell debris phagocytosed by macrophages.
RBC lysis →Hemoglobin from lysed RBC is catabolized by lysosomal protease→ release
of iron oxide to form insoluble aggregates→ with accumulation of iron ion in
Hemosiderin due to iron overload (secondary protective mechanism).

Persian blue iron stain on spleen Intra-alveolar deposition of hemosiderin
Hemosiderin accumulation in Scurvy

Deposition of hemosiderin in skin in severe scurvy
• Skin biopsy :deposition of hemosiderin is evident near hair follicles in patients
with severe scurvy .
Finegranularand
clumpedironof
hemosiderinstained
withPrussianblue . Bruise and hemorrhage in severe Scurvy

Therapeutic use of Vitamin C
❖Vitamin C:
a. can decrease the duration of cold episodes and severity of symptoms .
b. enhances the synthesis of immunoglobulins and increases the phagocytic
action of leucocytes.
c. may act by reacting with free radicals released by phagocytic leucocytes
(which become activated in infection) and decreases the inflammatory
effects(cytokine storm) caused by these oxidants . Therefore vitamin C used
as an adjuvant in infections (e.g.Covid19) .
d. has beneficial effects in the treatment of tuberculosis. Plasma level is kept
near saturation. Clinical dose is 500 mg/day.
e. is recommended for treatment of ulcer ,trauma/injury and burns.
✓Except scurvy and sub-scorbutic conditions, the therapeutic use of vitamin is
not specific.

Role of ascorbic acid in enhancement of immunity

The possible Beneficial effects of Ascorbic acid in
management of Covid -19(?)

Toxicity of vitamin C
❖Ascorbicacidassuch,hasnotbeenfoundtobetoxicandiswelltoleratedbyhealthy
subjects.
❖VitaminCbeingwatersoluble,Itisnotaccumulatedinthebody(vitaminoverload
unlikely).Itisexcretedassuch,althoughasmallportionisoxidizedtoDehydroascorbicacid
andthentooxalate.
❖Morethan2000mg(2000-3000mg)ofvitaminC/daypreventsinfections.But longterm
useofitsmegadose,cancauseironoverload(becausevitaminChelpsinabsorptionof
iron)withundesirableeffects.
❖Dehydroascorbic acid (oxidized form of ascorbic acid) is toxic.

PotentialbutrareadverseeffectsofMegadoseofvitaminC
1. cause severe gastrointestinal irritation (including nausea and diarrhea).
2. Increased oxalate excretion : Calcium salt of oxalates is major substance in
kidney stones. Calcium oxalate has been implicated in the formation of
kidney/urinary stones. There are controversial reports on megadose of
vitamin C leading to urinary calcium oxalate stones.
3. increased uric acid excretion(aggravates gout).
4. excess iron absorption(iron overload).
5. lowers vitamin B12 levels.
6. Pro-oxidant effects in presence in the presence of free Fe 3+ or Cu 2+ .
7. Systemic conditioning.
8. Rebound ‘Scurvy” .

Megadose of Ascorbic acid and its controversy
• Linus Pausing (Nobel laurate 1970) first advocated the consumption of
megadose of ascorbic acid up to 18g/day (300 times the daily requirement) to
prevent and cure common cold/ infections.
• Keep vitamin C in gunny bags and eat in grams.
• It is now clear that megadose of vitamin C dose not prevent common cold . But
the duration and severity of symptoms of cold are reduced.
• It is believed that ascorbic acid promotes leucocyte functions.
• Megadose (1-5 g /day) of vitamin C are still continued in common cold ,wound
healing ,trauma etc. and provides some health benefits.
• Quick-aging process is delayed.
• There are controversial reports on megadose of vitamin C leading to urinary
calcium oxalate stones.

Summary of functions of Vitamin C

Symptoms and management of Scurvy

Vitamin C (Ascorbic acid)

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