vitamin c( Ascorbic acid)

1. VITAMIN C

2. Vitamin C
• Scurvy: Sore gums, painful joints and
hemorrhages.
• Described by: Eber papyrus (1150 B.C.) and
Hippocrates (420 B.C.)
• Prevalence mainly among seamen:
– 1498: Portuguese, Vasco da Gama lost 60% of
crew
– 1535: French, J Cartier, most crew developed
scurvy

3. Chemistry
• L-ascorbic acid (MW 176) and its oxidized
derivative L-dehydroacorbic acid.
• They form a reversible redox system
• Quencher of free radicals, reduce iron and
other metals and superoxide radical.
• DHAA react with several amino acids to
form brown color
• Stable in acidic condition

4. VITAMIN C
OH
OO
H
HO
HO
HO

5. Sources
• Fruits, vegetables and organ meats (liver,
kidney)
• Loss due to oxidation, in the presence of
O2, heat, metal ions, neutral and alkaline
conditions.
• Cooking, loss due to heating and water
• Quick heating can protect by inactivating of
oxidases

6. Synthesis
• All Plants can synthesize Vitamin C
• Most animals can synthesize vitamin C
except : Humans, Guinea pigs, red-vented
Bulbul, fruit eating bat, rainbow trout, Coho
salmon
• They lack: L-gulono-γ-lactone oxidase

7. The biosynthesis of L-ascorbic acid.
α-D-glucose
glucose-6-phosphate
uridine diphosphate glucose
uridine diphosphate glucuronic acid
D-glucuronic acid-1-phosphate
D-glucuronic acid
NADPH·H+
L-gulonate pentose phosphate
NADP pathway
L-gulono-γ-lactone
O2
H2O2 L-gulono-γ-lactone oxidase
2-keto-L-gulonolactone
spontaneous isomerization
L-ascorbic acid

8. Absorption
• Occurs primarily by active transport
– Saturable and dose dependent
• Simple diffusion and carrier-mediated
contribute to small extent
• Prior to absorption, ascorbic acid may be
oxidized to dehydroascorbate
• Within enterocytes, dehydroascorbate
reduced to ascorbic acid
– Dehydroascorbate reductase
• Requires reduced GSH

9. Absorption
• Occurs primarily in distal portion of SI
– % absorbed decreases with increased vitamin intake.
• 16% at high intakes (6 g) and 98% at low intakes (<
20mg)
• Over range of 20 to 12 mg/d, get 80-95%
absorption
• Potential Factors Impairing Absorption
– Pectin (14.2 g/d)
– Zinc (9.3 mg/d)
– High iron content in GI can lead to destruction of AA

10. Absorption
• 200mg/day is a particular upper limit:
give 12mg/L plasma level
• 2,500mg/day: increase plasma level to
15mg/L
• Actively absorbed
–98% efficiency at 20mg/day
–16% efficiency at ~12g/day

11. Absorption and transport
• Absorption: Active transport system, Na
and ATP dependent.
• Efficiency of absorption decline with dose
above 1g.
• Transport in plasma as ascorbic acid
• Human cells become saturated at
100mg/day.
• Cellular uptake by Active transport system,
Na and ATP dependent.
• Uptake: glucose inhibits, insulin stimulates

12. Vitamin C
• Transport across basolateral membrane
– Sodium-independent carrier-mediated
transport
• Transported in plasma in free form
– Albumin may also transport some ascorbate
and dehydroascorbate (5% seen in circulation)
• Ascorbate moves freely into cells
– Concentration of ascorbate however is much
higher in some tissues
• Adrenal gland, pituitary gland and eye
– May be actively transported into these tissues

13. Ascorbic Acid
• Dehydroascorbic acid
– Taken up by red blood cells, lymphocytes and
neutrophils
• Reduced to ascorbic acid within cells
• Tissue and plasma level reflect intake until
intake exceeds ~90mg/day.

14. Tissue distribution
• In vital organs with active metabolism
• Total body pool size about 1500mg
• Half life about 20 days
• Turnover rate 1mg/kg/day
• Daily utilization breakdown is 0.2mg/kg fat
free weight

15. Metabolism
• Occurs primarily in liver
• Vitamin C is oxidized (removal of 2 electrons
and 2 protons) to dehydroascorbate
– Follows the formation of
semidehydroascorbate radical.
– Oxidized form may be reduced back to
ascorbate by GSH, NADH or NADPH.
– Oxidized form may be further oxidized to 2,3-
diketogulonic acid.
• Diketolulonic acid is cleaved into oxalic acid and 4
or 5 carbon sugars.

16. Urinary Excretion
• Oxidation: ascorbic acid, mono-dehydro-ascorbic
acid, dehydro-ascorbic acid.
• Excretion: 20-25 % ascorbic acid and DHAA, 20%
diketogulonic acid, 40-45% oxalate
• Dehydroascorbate, diketoglulonate, oxalic acid
and excess ascorbate excreted in urine
– 25% of vitamin C intake is excreted as oxalic acid
– Amount of Vitamin C filtered and then reabsorbed by
kidneys depends on plasma vitamin C concentrations
– Plasma levels above 1.4 mg/dL exceeds renal threshold
and vitamin C will not be reabsorbed.

17. Degradation of ascorbic acid
-2H
Ascorbic acid Dehydroascorbic acid
+2H
+H2O
Ascorbate-2-sulphate Diketogulonic acid
-C02
+ H2O Lyxonic acid
+O
+ H2O
Oxalic acid + Threonic acid -C02
-CO2 + H2O
+2H Xylonic acid
Xylose

18. Excretion
• Excretion reduced when intake is low
• Urinary excretion
–Body pool <1,500mg leads to only
metabolites in urine
–Body pool >1500mg leads to
proportionately more ascorbate in the
urine (can mask clinical tests)

19. Interactions With Other Nutrients
• Vitamin C increases intestinal absorption of
nonheme iron
– Reduces Fe3+
to Fe 2+
or forms a soluble complex
with the iron
• Excessive iron in presence of vitamin C can
accelerate the oxidative catabolism of
vitamin C
• Vitamin C aids incorporation of iron into
ferritin

20. Interactions with Other Nutrients
• Vitamin C may increase absorption and
excretion of heavy metals
– Form chelates with metals
• Vitamin C intakes above 600 mg/d may
interfere with copper metabolism
• Vitamin C helps keep folate in its reduced and
active form.

21. Metabolic functions
• Electron transport
• Antioxidant functions
• Prooxidant properties
• Enzyme cosubstrate functions:
– Collagen synthesis
– Neurotransmitter metabolism
– Carnitine synthesis
– Drug and steroid metabolism
– Tyrosine metabolism

22. Metabolic functions
• Metal ion metabolism
• Antihistamine reactions
• Health effects:
– Immune function
– Wound healing
– Cardiovascular disease
– Diabetes, cataracts
– Pulmonary function, Cancer

23. Vitamin C
Functions and Mechanisms of Action
• Antioxidant and Pro-oxidant Activity
– Reducing agent (antioxidant) (AH-
)
• Donate electrons and hydrogen ions
• AH-
may react with free radicals and reactive oxygen
species
– Reactive oxygen species
• OH (hydroxy radical), O2
-
(superoxide radical), H2O2
(hydrogen peroxide), and HO2 (hydroperoxyl radical)
• Attack phospholipids and protein embedded in
membranes
• Oxidize LDL and red blood cells

24. Antioxidant
Ascorbate (AH-) + OH
.
semidehydroascorbate radical (A-) + H2O
AH- + O2- + H+ A- + H2O2
AH- + H2O2 A- + H2O

25. Regeneration of Ascorbate
2 semidehydroascorbate radicals
ascorbate + dehydroascorbate
2 semidehydroascorbate radicals (A-) + 2
GSH
2 ascorbate (AH-) + GSSG
Dehydroascorbate (A) + 2 GSH
ascorbate (AH-) + GSSG
2 semidehydroascorbate (A-) + NADH + H+
2 ascorbate (AH-) + NAD+

26. Interconvertibility of ascorbic acid by oxidation and
reduction
ascorbate
[O] oxidase [H20]
Ascorbic acid Dehydroascorbic acid
GSSG glutathione 2 GSH
dehydrogenase

27. Vitamin C as Pro-oxidant
Ascorbate (AH-) + Fe+3
or Cu+2
semideydroascorbate radical (A-) + Fe+2
or Cu+1
The products Fe+2
and Cu+1
can proceed to cause cell damage
generation of reactive oxygen species and free radicals.
Fe+2
or Cu+1
+ H2O2
Fe+3
or Cu+2
+OH-
+ OH
.
Fe+2
or Cu+1
+ O2
Fe+3
or Cu+2
+ O2
-

28. Collagen Synthesis
• Most abundant protein found in body
– Major component of most connective tissue
• Skin, bone, cartilage, tendons, ligaments
• All collagen (n~19) have a triple helical structure
• For the collagen molecule to aggregate into its triple-helix
configuration selected proline residues must be
hydroxylated forming hydroxyproline
– Requires di-oxygenase enzymes, alpha KG, reduced
iron (Fe+2), ascorbate
– Ascorbate functions to reduce iron (cofactor) back to
its ferrous state.

29. O C
CH
Proline
CH2
CH2
HC
CH2
N
HN
NH2(CH2)4
Lysine
(CH2)2
COOH
CO2
α-ketogutarate
O C
COOH
OC (CH2)2
COOH
COO*H
Succinate
O C
CH
Hydroxyproline
CH
CH2
HC
CH2
N
HN
(CH2)4
Hydroxylysine
O C
OH
CH2 NH2CH
Dehydro-
ascorbate
Fe+3O2
Ascorbate
Fe+2
Ascorbate functions in the
hydroxylation of peptide-
bound proline and lysine in
procollagen.
*OH

30. Collagen Synthesis
• Vitamin C also required for hydroxylation of
lysine residues
• Hydroxylysyl residues permit cross-linking or
collagen and other post-translational
modifications
• Vitamin C may also influence mRNA levels
needed for collagen synthesis.

31. Carnitine Synthesis
• Vitamin C required for 2 reactions in the
synthesis of carnitine from trimethyllysine
– Trimethyllysine conversion to 3 hydroxy-
trimethyllysine requires
• Trimethylhydroxylase (dioxygenase), alpha KG, Fe+2
and
ascorbate
– 4-butyrobetaine to carnitine requires
• 4-butyrobetaine hydroxlyase (dioxygenase), alpha KG,
Fe+2
and ascorbate

32. (CH2)2
COOH
CO2
α-ketogutarate
COOH
OC (CH2)2
COOH
COOH
Succinate
Dehydro-
ascorbate
Fe+3O2
Ascorbate
Fe+2
Trimethyl
lysine
hydroxylase
CH2
CH2
CH2
CH2
H3C CH3
CH3
+NH3
COO-
+N
CH
Trimethyl lysine
CH2
CH2
CH2
HC – OH
H3C CH3
CH3
+NH3
COO-
+N
CH
3-OH-Trimethyl
lysine
The function of vitamin C in carnitine synthesis (1)

33. CH2
CH2
CH2
HC – OH
H3C CH3
CH3
+N
3-OH-Trimethyl
lysine
+NH3
COO-
CH
4-butyrobetaine
aldehyde
Glycine
+NH3
COO-
CH2
CH2
CH2
CH2
HC
H3C CH3
CH3
+N
O
NAD+
NADH
4-butyrobetaine
α-ketogutarate
Succinate
CO2
Dehydro-
ascorbate
Fe+3
O2
Ascorbate
Fe+2
4-butyrobetaine
hydroxylase
CH2
HC – OH
CH2
COO-
H3C CH3
CH3
+N
Carnitine
Serine hydroxymethyl
transferase-PLP-dependent
The function of
vitamin C in carnitine
synthesis (2)

34. Steps in carnitine biosynthesis
Lysine 6-N-Trimethyl lysine
6-N-Trimethyllysine Hydroxylase
3-Hydroxy-6-N-Trimethyl lysine
Glycine
γ-Butyrobetaine Carnitine
γ-Butyrobetaine Hydroxylase
Carnitine

35. Tyrosine Synthesis
• Hydroxylation of Phenylalanine
– Requires phenylalanine mono-oxygenase
(hydroxylase), Fe+2
, O2, tetrahydrobiopterin,
NADPH, vitamin C
• reducing power is supplied ultimately by NADPH but
immediately by tetrahydropterin
• Vitamin C may function in regeneration of
tetrahydrobiopterin from dihydrobiopterin.

36. CH2 – CH – COO-
+NH3
CH2 – CH – COO-
+NH3
CH2
O
CH2 – CH – COO-
+NH3
HO
O2 H2O
Tetrahydro-
biopterin
Dihydro-
biopterin
NAD(P)+ NAD(P)H
*
Phenylalanine hydroxylase Fe2+
COO-
C
HO
H2O
O2
Dihydro-
biopterin
Tetrahydro-
biopretin
NAD(P)H
NAD(P)+
Tyrosine mono-oxygenase/hydroxylase-Fe2+
Phenylalanine Tyrosine
α-keto-
glutarate
glutamate
Tranaminase
-vitamin B6-
dependent
HO
HO 3,4-dihydroxyphenylalanine
(DOPA) P-hydroxyphenylpyruvate
The role of vitamin C*
in the phenylalanine
and tyrosine
metabolism, including
norepinephrine
synthesis

37. CH2 – CH – COO-
+NH3
HO
HO
3,4-dihydroxyphenylalanine
(DOPA)
CO2
DOPAdecarboxylase – Vitamin B6
(CH2)2 – NH2HO
HO
Dopamine
O2
H2O Cu2+
Cu1+
Dopamine
mono-
oxygenase
Dehydro-
ascorbate
Ascorbate*
CH – CH2 – NH2HO
HO
OH
Norepinephrine
The role of vitamin C* in the
phenylalanine and tyrosine
metabolism, including
norepinephrine synthesis

38. O2 CO2
Cu2+ Cu1+
P-hydroxy-
phenylpyruvate
hydroxylase
Dehydro-
ascorbate
CH2
O
COO-
C
HO
P-hydroxyphenylpyruvate
Ascorbate*
OH
CH2 – COO-
HO
Homogentisate
O2
Cu2+
Cu1+
P-hydroxy-
phenylpyruvate
hydroxylase
Dehydro-
ascorbate
Ascorbate*
-OOC – CH = CH – C – CH2 – C – CH2 – COO-
OO
4-maleylacetoacetate
The role of vitamin C* in
the phenylalanine and
tyrosine metabolism,
including norepinephrine
synthesis

39. Tyrosine Catabolism
Tyrosine
p-Hydroxyphenylpyruvic acid
*
Homogentisic Acid
*
Maleylacetoacetic Acid
Fumarylacetoacetic Acid
Acetoacetic Acid
Acetoacetyl CoA
p-hydroxypenylpyruvate dioxygenase
Homogentisate dioxygenase
Ascorbate, Cu2+
Ascorbate, Fe2+

40. Tyrosine Metabolism
Tyrosine
3,4-dihydroxyphenylalanine
(Dopa)
Dopamine
Norepinephrine
Cu1+
Vitamin C
Dopamine
Mono-oxygenase

41. Amidation of Peptides with C-
terminal glycine (hormone activation)
• Peptidylglycine amidating oxygenase
– Requires Cu+1
, ascorbate, O2
– Functions to cleave the carboxyl-terminal
through use of molecular O2.
• Amino group is retained as terminal amide while
rest is released as glyoxylate
• Many of amidated peptides resulting from this
reaction are active as hormones, hormone-releasing
factors and neurotransmitters
– E.g.Gastrin, CCK, oxytocin, corticotropin, calcitonin,
thyrotropin, vasopressin

42. Proline HO-Proline Dopamine Norepinephrine
Proline Dopamine
Monooxygenase Monooxygenase
Semihydroascorbate Ascorbate Semidehydroascorbate Ascorbate
Ascorbate Semihydrosascorbate
O Amidating O O
(Inactive Hormone)R-C-N-CH2COOH Enzyme R-C-NH2+HC-COOH (Amidated Hormone)
H
Fe2+
Fe3+
Cu+
Cu2+

43. Serotonin Synthesis
• Serotonin can be synthesized from tryptophan
• Hydroxylation of Tryptophan to 5-hydroTrp
– Requires tryptophan mono-oxygenase
• O2, tetrahydrobiopterin, vitamin C and NADPH
• Decarboxylation of 5-OHTrp to Serotonin

44. Serotonin synthesis
Tryptophan mono-oxygenase-Fe2+
O2 H20
CO2
Tetrahydrobiopterin Dihydrobiopterin
(5-hydroxytryptamine)
*
NADP+
NADPH+H+
*Vitamin C may function in tetrahydrobiopterin regeneration
TryptophanTryptophan 5-hydroxytryptophan5-hydroxytryptophan
Serotonin

45. Enzymatic reactions involving ascorbic acid-
dependent dioxygenases
Reaction Substrate Enzyme Product
1
2
3
4
5
6
p-OH-phenylpyruvate
hydroxylase1
Peptidyl L-proline
Peptidyl L-proline
Peptidyl L-lysine
6-N-Trimethyl L-lysine
4-N-Trimethyl
aminobutyrate
p-OH-phenylpyruvate
Prolyl 4-hydroxylase2
Prolyl 3-hydroxylase2
Lysyl hydroxylase2
6-N-Trimethyl L-lysine
hydroxylase2
4-N-Trimethyl amino-butyrate
hydroxylase2
Homogentisate
Peptidyl 4- transhydroxyl-L-
proline
Peptidyl 3- transhydroxyl-L-
proline
Peptidyl5-erythrohydroxy- L-
lysine
Erythro-3-hydroxy-6-N-
trimethyl –L-lysine
3-Hydroxy-4-N-trimethyl
amino-butyrate
1
α-Ketoglutarate is not required as a cosubstrate.
2
α-Ketoglutarate is required as a cosubstrate
Source: modified from Englard and Seifter (1986)

46. Vitamin C
• Vitamin C and The Common Cold
– 1 g Vit C /d = 50mg/d
• Number of colds, severity and duration
– 1 g Vit C/d decreases duration and severity of
symptoms
• Vitamin C and Cancer
– Controversial
• Epidemiologic studies suggest inverse relationship
between Vitamin C and cancers of oral cavity,
esophagus, and uterine cervix

47. Vitamin C and Cancer
• Clinical Studies
– Some researchers have shown that survival
time in cancer patients may be prolonged;
– Others have not shown this
• Possible Protective Mechanisms
– Ability to act as a reducing agent
– Detoxify carcinogens
• Vitamin C ingested with nitrates or nitrites can
prevent formation of nitrosamines or nitrosamides

48. Vitamin C
• Atherosclerosis:
– Negative relation with vitamin C
– LDL lipid peroxidation
• Cataracts
– Negative relation with vitamin C
• Bone density
– Positive relation with vitamin C
• Wound healing and connective tissue
metabolism

49. Recommended dietaRy
allowances (Rda)
For adults: 60-70mg/day
For pregnancy & lactation: 20-40%
more than the normal RDA

50. Deficiency
• Vitamin C intakes < 10 mg/d result in scurvy
– See when total body pool is < 300 mg
– Symptoms
• bleeding gums, small skin discoloration due to
ruptured blood vessels, easy bruising, impaired
wound and fracture healing, joint pain, loose and
decaying teeth, hyperketatosis of hair follicles.
• Scurvy rare in US
– Low plasma vitamin C levels observed in
elderly

51. Vitamin C Deficiency
Corkscrew Hair

52. Vitamin C Deficiency
Perifollicular Petechiae

53. Vitamin C Deficiency
Scurvy

54. Vitamin C (Ascorbic acid)
Gum changes in infant
scurvy:
The swelling and
hemorrhages are confined to
the areas of the gum
surrounding the erupting
teeth.

55. Vitamin C (Ascorbic acid)
Gums in scurvy:
The gums are blue-red and glossy
swollen in this patient with severe
scurvy. The earliest changes are
swelling of the internal dental
papillae and tendency to bleed
easily. Lesions occur only in
relation to teeth and so in young
infants and edentulous adults they
are absent. In advanced cases
there is usually an element of
infection and antibiotics as well as
vitamin C are required for healing

56. Vitamin C (Ascorbic acid)
Very advanced gum lesions in scurvy

57. Vitamin C (Ascorbic acid)
Orbital hemorrhage:
This is a dramatic but infrequent
sign of scurvy. There is complete
clearing with treatment.

58. Vitamin C (Ascorbic acid)
Splinter hemorrhage:
In this unusual sign in scurvy the
hemorrhages are arranged in a
semicircular lattice involving nail
beds. They are more extensive
than those in sub-acute bacterial
endocarditis.

59. Vitamin C (Ascorbic acid)
Perifillicular petechiae:
Minimal bleeding into the hair follicles is
pathognomonic of vitamin C deficiency
and is often the earliest clinical
manifestation. In vitamin K deficiency,
thrombocytopenia and other conditions,
petechiae are situated in areas of skin
unrelated to the hair follicles. In
perifillicular hyperkeratosis, there is no
bleeding and hyperkeratosis is present.
Ecchymoses develop in more advanced
deficiency and are the most frequent sign
in “workhouse” scurvy in old men. Wound
healing is markedly delayed.

60. Scurvy
• After 45-80 days of stopping vitamin C intake
• The 4Hs:
– Hemorrhagic signs
– Hyperkeratosis of hair follicles
– Hypochondriasis (Psychological)
– Hematological (impaired iron absorption)

61. Toxicity
• Toxicity more likely with ingestion of
several large (1g) doses than one single
dose
– Remember absorption is saturable and dose
dependent
– Kidney stones?
• oxalic acid + calcium make up kidney stones
– People predisposed to kidney stones should avoid high
intakes
– Urate crystals and urate kidney stones
• Vitamin C competes with uric acid reabsorption

62. Toxicity
• Toxicity is rare
• Increased intake and B6 and B12 utilization
• Chronic high doses of vitamin C may be
unsafe for those unable to regulate
absorption of iron
– Hemochromatosis
• May interfere with clinical tests
– Tests for glucose in urine
• Decrease intake gradually to avoid scurvy-
like symptoms

63. Assessment of Nutriture
• Blood, serum or plasma levels most
commonly used.
• Change in response to recent dietary
Vitamin C intakes
• Plasma or serum levels most sensitive
indicators of deficiency
• White blood cell content reflects body
stores
– Measurement is technically difficult.

64. Pharmacological uses
• Megadoses for fighting infections
• Vitamin C and common Cold.
• Vitamin C and stress.. chickens