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Vitamins
58.2. Vitamins soluble in water
2
58.2.1. Thiamine
H3C
S
N
HO
+
134
5 2CH3
1 2
45 N
N
NH2
Thiamine, Vitamin B1
3-(4-Amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-
methylothiazolium chloride
A molecule of thiamine consists of a substituted thiazol ring attached
to a substituted pyrimidine ring by a methylene bridge. The
substituents in positions 2 and 4 in the pyrimidine ring and 4 and
5 in the thiazol ring are very important for the activity of
thiamine.
The replacement of the methyl group by an ethyl group in the
position 2 of pyrimidine does not have much influence on activity,
whereas the C2 butyl derivative acts antagonistically.
3
The acetylation of the amino group in position 4 in the
pyrimidine ring weakens action, while the replacement of the
amino group by a hydroxyl group creates oxythiamine with
antagonistic action (antivitamin B1).
The hydroxyethyl group in the position 5 of the thiazol ring is
very important for the activity of thiamine. Its elimination or
replacement by another substituent results in the disappearance
of activity.
The replacement of the hydrogen atom in the position 2 of the
thiazol ring by a sulfur atom (Thiothiamine, vitamin S-B1) does
not influence activity.
In therapy thiamine analogues with an ‘open’ thiazol ring
(acetiamine, benfotiamine, fursultiamine, prosultiamine) which
act similarly to thiamine, are also used.
4
Some of their properties are even better than those of thiamine, for
example lower toxicity, higher stability, prolonged action and better
absorption from the GT. In the body, they are a source of vitamin B1
and are used as analgetic agents.
H3C
CH3S
N
S
NH2
N
NHO
Thiothiamine, Vitamin S-B1, SULBONE
CH3
CH3NH2
N
N
NO
SCHO
PO3H2
O
Benfotiamine, BIOTAMIN
H3C
H3C
NH2CH3
CH3
O
OCHO
N
N
N
S
O
Acetiamine, THIANEURON
CH3
CH3 NH2
R
CHO
N
N
N
SS
HO
Prosultiamine, R = -CH2-CH2-CH3; DITIAMINA
Fursultiamine, DiavitanR =
O
5
The daily requirement of witamin B1 depends on a person’s age (low
in children, 0.3–1.0 mg/24 h) and sex (slightly higher in men – 1.2–
1.5 mg/24 h – than in women – approx. 1.1 mg/24 h). It also depends
on nutrition (greater in the case of a diet rich in carbohydrates) and it
is higher in pregnant women in (1.5 mg/24 h) and during lactation
(1.6 mg/24 h).
A deficiency of vitamin B1 increases the concentration of pyruvic
acid in the tissues and leads to the beriberi disease, which is
characterized by the disturbance of the nervous system (peripheral
sensomotor polyneuropathy and encephalopathy) with cardiac
insufficiency, edemas and psychic disturbances. In vitamin B1
deficiency medicinal products containing synthetic thiamine are used.
They are administered mostly orally in daily doses of 3–9 mg and in
serious cases intramuscularly, 10–20 mg once or twice daily.
6
Thiamine is needed for the metabolism of carbohydrates. It is also
vital for the supply of energy necessary for the function of the
nervous system, the cardiac muscle and skeletal muscles. In the body
thiamine is phosphorylated to active thiamine pirophosphate (TPP)
and thiamine triphosphate (TTP).
TPP is a coenzyme for pyruvate decarboxylase, 2-ketoglutarate
dehydrogenase and transketolase. TPP participates in the transfer of
the aldehyde group in the pentose phosphate cycle.
After oral administration the transport of vitamin B1 depends on the
dose. At concentrations < 2 mol active transport by a carrier occurs,
while at concentrations >2 mol passive diffusion is observed. The
absorption of thiamine is the greatest in the duodenum and the upper
and middle sections of the small intestine. Thiamine is not absorbed
from the stomach or the distal fragment of the small intestine.
Thiamine is absorbed after its phosphorylation in the epithelium.
7
Vitamin B1, after oral administration, is not absorbed completely. Its
maximal absorption is 8–15 mg/24 h. The absorption of thiamine is
greater when it is administered in divided doses during meals. An
excess of administered thiamine is eliminated in urine. The half-time
of the -phase elimination of thiamine is 1.0 h. Thiamine is
eliminated unchanged and as metabolites – thiaminecarboxylic acid,
pyramine and others which have not been identified yet.
8
The stability of thiamine
The greatest stability is demonstrated by solutions of thiamine at pH
2, which can be stored for 6 months at 37 0C, without any effect on
activity. Rapid degradation is observed at pH > 5, especially in the
presence of atmospheric oxygen. The aqueous solutions at pH < 5
can be sterilized for1 h at 100 0C. Chlorobutanol at a concentration
of 0.5% is recommended for the stabilization of thiamine chloride in
some pharmacopeal monographs.
The acidic hydrolysis of thiamine splits the bond N-C between the
nitrogen atom of the thiazol ring and the carbon atom of the
methylene group. In an alkaline environment a thiol form of
thiamine is formed, which is very reactive and produces a
benzodiazepine derivative after the elimination of hydrogen sulfide.
In the presence of oxygen hydrogen sulfide is oxidized to elemental
sulfur insoluble in water.
9
NH2
CH3
CH3
THIAMINE
4-Amino-5-hydroxy-
methyl-2-methyl-
pyrimidinenvironment
basic and
neutral
H
thiol form of thiamine
N
N
N
SCHO
H+ /H2O
H3C
S
N
HO
+
5-(2-Hydroxyethyl)-
4-methylthiazol
NH2
CH3
HO N
N
HO
O2
H2S
S
CH3
H3C
H
NH2
N
N
N
N
HO
Diazepine derivative
Figure 58.5.
The degradation of thiamine in acidic, basic and neutral
environments.
10
58.2.2. Riboflavin
H3C
H3C
O
ON
NN
N
H2
C
C
C
C
CH2OH
H
H
H
HO
HO
HO
H
Riboflavin, VITAMINUM B2
7,8-Dimethyl-10-(D-1-ribityl)-isoalloxazine
The term riboflavin is made up of the names of its two components
- ribitole and flavin. The word flavin is derived from flavus, the
Latin word for yellow. The following relationship between the
chemical structure and action of riboflavin is observed:
11
For biological activity the presence of two methyl groups in
positions 7 and 8 is necessary; when they are absent or their
position is changed action disappears. The replacement of the
methyl groups by a chlorine atom produces compounds with
antagonistic action.
A change of ribitol’s configuration from D to L causes action to
disappear.
The imine group (-NH-) in position 3 is necessary for activity.
When the hydrogen atom is replaced by a methyl group in the
imino group action disappears.
Riboflavin is a precursor of riboflavine-5'-phosphate (flavin
mononucleotide; FMN) and flavin adenine dinucleotide (FAD).
The names nucleotide and dinucleotide are not correct because
ribitol is not a pentose and is not fused to flavin with a glycoside
bond but these terms are accepted because of their widespread
use.
12
H3C
H3C
H3C
H3C
H3C
H3C
FAD = Oxidized form
Reduced form
FADH2
Free radical (FADH)
.H
2H .
H.
N
NN
N
R
H
O
O
H
.
N
NN
N
R
H
O
O
H
H
N
NN
N
R
H
O
O
Enzymes containing riboflavin are called flavoproteins. They
participate in oxidation-reduction reactions. Flavin coenzymes can
exist in any of 3 different red-ox states: oxidised flavin (FAD),
semiquinone (free adical) (FADH) or reduced (FADH2) flavin.
13
FAD is converted to semiquinone by one-electron transfer. A second
one-electron transfer converts semiquinone to the completely reduced
dihydroflavin.
The presence of the three red-ox forms makes it possible for flavin
coenzymes to participate in one- or two-electron transfers.
Because of that flavoproteins calalyse many biochemical reactions
together with various acceptors and donors of electrons such as:
transmitters of 2 electrons (NAD+, NADP+, DT-diaphorase)
one- and two-electron transmitters e.g. quinones
one-electron transmitters (cytochrome proteins).
14
Examples of flavoproteins include:
dehydrogenases (acylo-CoA dehydrogenase, glutathione
reductase, aldehyde dehydrogenase, mitochondrial glycerol-3-
phosphate dehydrogenase, succinate dehydrogenase of the citric
acid cycle, NADH dehydrogenase of the respiratory chain in
mitochondria, dihydrolipoyl dehydrogenase)
monooxygenases (lactate oxygenase)
dioxygenases
oxidases (glucose oxidase, -amino acid oxidase, xanthine
oxidase).
15
Vitamin B2 exists in many foods. Sources of this vitamin are liver,
white meat, eggs, milk and fresh vegetables. The daily requirement
of vitamin B2 is 1–3 mg. A deficiency of vitamin B2 inhibits growth
in children. Symptoms of hypovitaminosis are inflammation of oral
mucosa and the tongue, angular cheilitis (the cracking of mouth
corners), seborrheic dermatitis.
Vitamin B2 is used in the treatment of cataract and inflammations of
mucous membranes as well as in convalescence after devastating
diseases:
orally in doses of 3–9 mg daily
in severe deficits of vitamin B2, 5–10 mg daily (i.m.).
16
The stability of riboflavin
When stored in the solid state and protected from light riboflavin is
stable, even at an increased temperature. Under the influence of light
and oxygen it is decomposed to lumiflavin. This reaction occurs
easily in an alkaline environment under the influence of visible
radiation. In neutral and acidic solutions, under the influence of light,
lumichrome consisting of various lumiflavins is formed.
H3C
H3C
N
NN
N
H
O
O
H
Lumiflavin
Lumichrom
+
H3C
H3C
CH3
Lumiflavin
N
NN
N
H
O
O
N
NN
N
R
H
O
O
Riboflavin
hv/H+
H3C
H3Chv/OH-
Figure 58.6.
The decomposition of
riboflavin under the influence
of light.
17
58.2.3. Pantothenic acid
H3C
H3CN
O
COOH
OH
HO
HPantothenic acid, Vitamin B5
(R)-N-(2,4-Dihydroxy-3,3-dimethyl-1-oxobutyl)--alanine.
A molecule of pantothenic acid consists of -alanine, which is fused
to 2,4-dihydroxy-3,3-dimethylobutanoic acid (pantoinoic acid) with
an amide bond. Only a dextrorotatory R-isomer shows vitamin
activity. Similar action is shown by a product of the reduction of
pantothenic acid – dexpantenol, which is also used in therapy. In the
body dexpantenol is easily oxidized to pantothenic acid.
The introduction of a methyl group in position
(-methylpantothenic acid) or replacement of the carboxyl group by a
sulfonic (-SO3H; pantoilotaurin) or benzoyl (phenylpantothenone)
group creates compounds with antivitamin activity.
18
PANTOTHENOATE
4-Phosphopantothenoate
4-Phosphopantothenylcysteine
4-Phosphopantotein
Dephospho-CoA
Coenzyme A
ACP
ATP
ADP
ATP
ADP +
ATP
ATP
ADP
PPi
P
CO2
Serine
Cysteina
* Phosphorylation of pantothenoate
-
Ribozyl-3'-phosphate
Adenine
P O
O
O
O
P O
O
O
O
O-
O
O OHP
O
O -
CH2
NH2
N
N
N
N
H3C
H3C OH
CH C CH C N CH2 CH2 C N CH2 CH2 SH_ __
O_
P
O
O
O -
- __
O
__
O
_ __ _
Pantoinic acid -Alanine Mercaptoetanoamine
Pantothenic acid
* Binding with cysteine
* Decarboxylation of cysteine rest
hydroxyl group ( ACP)
( Dephospho-CoA)
or binding with cystein by
* Adenylation of 4-phospho-
pantetheine
3'-Phosphorylation*
H H
Pantothenic acid is a component
of coenzyme A (CoA) and acyl-
carrying protein (ACP).
Their biosynthesis consists of
reactions shown in Figure 58.7.
Figure 58.7.
The biosynthesis of coenzyme A
and ACP from pantothenic acid.
19
The SH group participates in the transfer of the acyl groups, while
adenine nucleotide acts as the recognition site increasing the affinity
and specificity of coenzymes for enzymes that bind with it.
Coenzyme A is a transporter of the acyl group in the citric acid
cycle, in the oxidation and synthesis of fatty acids and in the
reactions of acetylation of endogenic substances and drugs, e.g. in
the synthesis of acetylcholine and cholesterol. Coenzyme A
influences the function of the GT, the regeneration of the epithelium
and the growth of hair and nails.
The daily requirement of pantothenic acid is 8–10 mg. It is supplied
in a normal diet. Spontaneous pantothenic acid avitaminosis is not
observed because it exists commonly in nature and is synthesised by
certain intestinal bacteria. Pantothenic acid is found in yeast, liver,
yolk of eggs, legumes, grains of cereals, vegetables, milk and fruits.
20
Calcium pantothenate (CALCIUM PANTOTHENICUM) and
Dexpantenol (BEPANTHEN) are used in streptomycin poisoning, in
postoperative intestinal atonia, paralytic ileus, rheumatoidal diseases
and locally in the damage of the cornea, in skin, hair and nail
diseases, in pharyngitis, rhinitis, chronic and acute sinusitis, and as
supplement to foods and vitamin products.
Pantothenic acid for pharmaceutical products is obtained
synthetically.
The amide bond in pantothenic acid is sensitive to acidic and
alkaline hydrolysis.
21
58.2.4. Pyridoxine
CH365
4 3
21
R
OHHO
N
Pyridoxine, R = -CH2OH; VITAMINUM B6
3-Hydroxy-4,5-di(hydroxymethyl)-2-methyl-pyridin
Pyridoxal; R = -CHO
Pyridoxamine; R = -CH2NH2
In therapy pyridoxine is used as vitamine B6 but vitamin properties
are demonstrated by three pyridine derivatives: pyridoxine, pyridoxal
and pyridoxamine. In the body, they undergo reversible
transformation under the influence of enzymes:
pyridoxine pyridoxal pyridoxamine
22
Pyridoxal shows the greatest activity. The oxidation of the aldehyde
group to the carboxyl group eliminates activity. The replacement of
substituents at C4 and C5 by methyl or alkoxymethyl groups
produces compounds with antagonistic action
The active form of vitamin B6 is pyridoxal 5-phosphate (PALP),
which in the body exists in two tautomeric forms.
CH3N
OHOP
O
O
O
-
-
CHO
CH3+
-O
HN
OP
O
O
O
-
-
CHO
PALP is the coenzyme of many enzymes (transaminases,
decarboxylases, racemases and others), participating in
nonoxidative tansformation of amino acids.
23
Enzymes dependent on PALP participate in the following reactions:
transamination, leading to the degradation and synthesis of amino
acids
- and -decarboxylation, as a result of which biogenic amines
are formed: dopamine, noradrenaline, histamine, serotonine,
tyramine, GABA
- and -elimination, leading to the synthesis of ketoacids, e.g.
pyruvate is formed from serine by -elimination of ammonium
racemization
aldol reaction.
The intermediate product in these reactions is Schiff’s base.
24
The daily requirement of vitamin B6 is ~1.25 mg and is supplied in a
normal diet. Sources of vitamin B6 are yeast, rice bran, crop
germ, meat, celery, lettuce and peppers. A deficit of witamin B6
can be caused by wrong nutrition or the use of certain drugs, for
example isoniazid, hydralazine or penicillamine. These drugs
deactivate pyridoxal phosphate because together with PALP they
form Schiff’s base.
The following symptoms are observed in vitamin B6 deficiency:
decreased synthesis of serotonine and noradrenaline, which can
lead to neuropathy and depression
increased elimination of xanthurenic acid in urine (PALP is the
coenzyme of kinureninase).
The symptoms of B6 avitaminosis are nausea, vomiting, damage of
the skin and mucous membranes, psychical disturbances,
convulsions, polyneural inflammation and anemia.
In therapy synthetic pyridoxine is used.
25
Pyridoxine is applied:
in the treatment of convulsions in children caused by the
hereditary mutation of the apoenzyme of cerebral glutaminic acid
decarboxylase
in congenital anemia, caused by the genetic defect of the
apoenzyme of the synthesis of -aminolevulinic acid, whose
coenzyme is PALP
in the treatment of acrodynia (pain and inflammation of the skin
on the ends of the nose, hands and feet)
as an auxiliary drug in the treatment of changes of the skin and
mucosa, leucopenia and agranulocytosis
during convalescence after devastating diseases.
Pyridoxine is well absorbed from the GT. In the blood 80% of PALP
is bound with proteins. The main metabolite of pyridoxine is
inactive 4-pyridoxine acid.
26
58.2.5. Biotin
* ** OH
O
H H
HH
O
NN
S
12
3
456
Biotin, Vitamin B7, Vitamin H
cis-Tetrahydro-2-oxothiene-[3,4]imidazoline-4-valeric acid
Biotin is composed of an tetrahydroimidazolone ring fused with a
tetrahydrothiophene ring. A valeric acid subsituent is attached to
one of the carbon atoms of the tetrahydrothiophene ring.
In one molecule of biotin three asymmetric carbon atoms exist, so 8
optic isomers are possible. Additionally cis/trans isomerism is
possibile. Only cis D-biotin shows vitamin activity. The length of
the chain in the thiophene ring is also very important. The
shortening or elongating of this chain or introducing a double bond
into it produces compounds with antagonistic action.
27
Biotin exists in many natural foods. Its best sources are yolk of eggs,
yeast, liver and kidneys. Biotin is also synthesised by saprophytic
intestinal bacteria.
Biotin is a cofactor responsible for the transfer of the carboxylate
group in several carboxylase enzymes:
alfa and beta acetyl-CoA carboxylase
-methylcrotonyl-CoA carboxylase
propionyl-CoA carboxylase
pyruvate carboxylase.
28
NN
S
O
H H
HH
O
~1,5 nm
N
O
H
Biotin-lysine complex =
biocitine
Biotin is the prosthetic group of the enzyme with which it is bound
by the -amino group of the lysine rest.
A chain composed of 10 atoms separates the biotin ring from the
carbon atom of lysine to a distance of approx. ~1.5 nm. This chain
is responsible for the ability of biotin to transfer the carboxylate
group.
29
Biotin-dependent enzymes are carboxylases: pyruvate, acetyl-CoA,
propionyl-CoA, -methylcrotonyl-CoA. In most biotin-dependent
reactions the source of the carboxylate group is a hydrocarbonate
ion.
Large amounts of hydrocarbonate ions exist in biological fluids, but
because their electrophilicity is weak hydrocarbonate must be
activated first.
ATP and Mg(II) ions participate in the activation of hydrocarbonate
ions.
The carbonate-phosphate anhydride created in this reaction transfers
the carboxylate group to the N1 atom of the biotin ring.Next the
carboxylate group is transferred to the substrate. (Fig. 58.8).
30
32
Acetyl-CoA
Carboxybiocitine rest
Biocitine rest
P
Anhydride of phosphoric and carbonic acid
NN
S
O
H
R
O
O-
NN
S
O
H H
R
H3C
O
__ SCoAC
Malonyl-CoA
C SCoA_ _
O
-COO
H2C
1
P C
O
O O
O
O
O-
-
-
Mg2+
ATP
ADP
HCO3-
Figure 58.8.
The role of biotin in the carboxylation of acetyl-CoA.
– The activation of hydrocarbonate by ATP, – the carboxylation of biotin,
– the transcarboxylation (the transport of the carboxylate group from biotin to
the substrate).
31
Biotin as the coenzyme of carboxylases participates in the
biotransformation of:
pyruvate to oxalacetate, which is very important for
gluconeogenesis
acetyl-CoA to malonyl-CoA, which is an important substrate in
the synthesis of fatty acids
propionyl-CoA to methylmalonyl-CoA, which participates in the
citric acid cycle
-methylocrotonyl-CoA to -methylglutaryl-CoA, a precursor of
malonic acid, which is a substrate in the biosynthesis of steroids.
32
Biotin is absorbed from the small intestine as a result of active
transport and passive diffusion.
Aprox. 80% of biotin is bound with plasma proteins. The
concentration of free and bound biotin in the blood is 200–1200 ng/l.
The level of biotin in erythrocytes is only ~10% of its concentration
in plasma.
The elimination of biotin occurs in the kidneys and intestines. Its
half-time depends on dosage and for an oral dose of 100 g/kg of
body mass it is ~26 h.
This period is shorter (10–14 h) for the same dose in people with a
deficit of biotin.
The daily requirement of biotin is 10–30 g in children (depending
on age) and 30–100 g in adolescent and adults.
33
Biotin deficiency can be caused by:
excessive consumption of raw egg-whites over a long period
(months to years); egg-whites contain high amounts of avidin, a
protein that binds biotin strongly and irreversibly. The biotin-
avidin complex is not broken down or liberated during digestion,
but removed from the body in feces. When cooked, the egg-
white avidin becomes denaturated and entirely non-toxic;
the deficit of holocarboxylate synthase catalysing the attachment
of biotin to the lysine rest of carboxylate apoenzyme;
oral therapy using antibiotics and sulphonamides.
34
People with type 2 diabetes often have low levels of biotin.
Biotin may be involved in the synthesis and release of insulin.
Initial symptoms of biotin deficiency include: dry skin, seborrheic
dermatitis, fungal infections, rashes including erythematous
periorofacial macular rash, thin and brittle hair, hair loss or total
alopecia.
If left untreated, neurological symptoms can develop, including mild
depression, which may progress to profound weakness and,
eventually, to somnolence, changes in mental status, generalized
muscular pains (myalgias), hyperesthesias and paresthesias.
Biotin is most often used together with other vitamins of the B group
in psoriasis, seborrhea and dermatitis. In therapy biotin obtained
synthetically is used.
35
58.2.6 Cobalamins (Vitamins B12)The chemical structure of vitamins B12
is based on a corrin ring, which is
similar to the porphyrin found in heme,
chlorophyll, and cytochrome. The
central metal ion is cobalt. Four of the
six coordination sites are provided by
the corrin ring, and the fifth by a
dimethylbenzimidazole group. The
sixth coordination site, the center of
reactivity, is variable, being a cyano
group, a hydroxyl group, a methyl
group or a 5’-deoxyadenosyl group (the
C5’ atom of the deoxyribose forms the
covalent bond with Co).
CH3
CH3
N
N
O P O
O
O
N N
NN
N
Co+
R
H
HO
HOO
-
H3C
H3C
H3C O
O
O
O
O
O
O
CH3
CH3
CH3
CH3
CH3
CH3
H2N
H2N
H2N
NH2
NH2
NH2
R = -CN; Cyanocobalamin;
R = OH; Hydroxycobalamin
36
Vitamin B12 is naturally found in foods, including meat (especially
liver and shellfish), eggs and milk products.
Vitamins B12 are necessary for the biosynthesis of nucleinic acids.
Vitamins B12 are absorbed as a result of active transport. An internal
factor (glycoprotein secreted by parietal cells) is indispensable for the
absorption of vitamins B12.
The cobalamin-internal factor complex is transported to the ileum,
where it binds with the specific receptors in the epithelium of the
intestine. After the dissociation of this complex to cobalamin and the
internal factor, cobalamin is transported to mucous cells and from
them to the portal circulation. The amount of vitamin B12 that is
absorbed depends on the concentration of the internal factor, the
pancreas function and the density of receptors in the ileum. The
maximal concentration is observed 4–8 h after administration.
37
The maximal concentration is observed 4–8 h after administration.
The absorption of vitamin B12 is decreased by aminoglycoside
antibiotics, aminosalicylic acid, biguanids, chloramphenicol,
colestyramine, potassium salts, methyldopa and antiepileptic drugs.
Vitamin B12 is not absorbed in Addison’s disease.
After absorption, vitamin B12 is transported with the blood in a form
bound with -globuline – transcobalamin, and reaches the liver and
other tissues. In the body, vitamin B12 (inactive) is transformed to
two active coenzymes – 5'-deoxyadenosylcobalamin and
methylcobalamin. These coenzymes are formed in various
compartments – methylcobalamin in cytosol and adenosylcobalamin
in the mitochondria.
38
Homocysteine Methionine
Metionine synthase
Methylocobalamin
5-Me-THF THF
+
S
+
CH3
NH3NH3-OOC -OOC
HS
Methylcobalamin participates in the coupled conversion of:
homocysteine to methionine and
methylotetrahydrofolate (Me-THF) to tetrahydrofolate (THF).
39
In the lack or deficit of methylcobalamin
homocysteine accumulates in the body; homocysteine is a risk
factor in sclerosis
a deficiency of methionine is observed; methionine participates
as adenosylmethionine in the conversion of
phosphatydyletanolamine to lecitin in the cell membrane
etanolamine to choline, which is a precursor of ACh; a
deficiency of methionine may cause neurologic disturbances
a deficiency of THF is observed, which results in a decreased
synthesis of purine and pyrimidine bases and a defect of DNA
synthesis leading to anemia.
40
Deoxyadenosylcobalamin participates in the intramolecular
rearrangement of L-methylmalonyl-CoA to succinyl-CoA.
COO-H
CH3
C
CS CoAO
L-Malonyl-CoA
5'-Deoksyadenosylocobalamine
COO-
H
CH2
C
_
H
CoAOC
S
Succinyl-CoA
Methylmalonyl-CoA mutase
In a deficiency of vitamin B12 the rate of this transformation is
decreased and the level of propionyl-CoA and methylmalonyl-
CoA, which can be used in the synthesis of fatty acids, is
increased.
Vitamin B12 is slightly soluble in water and accumulated in the
liver.
41
Vitamin B12 deficiency can be caused by:
its insufficient amount in food (vegetarian diet)
deficiency of the internal factor (pernicious anemia, Addison’s
anemia, Biermer’s anemia)
intestinal diseases
congenital (inborn) deficiency of transcobalamin II
a disturbance of the intrahepatic circulation.
Vitamin B12 is administered:
orally, only to eliminate its deficiency in food and to people with
an increased requirement of this vitamin
intramuscularly or subcutaneously to people with a high
deficiency and megaloblastic anemia and with neurological
symptoms.
42
58.2.7. Nicotinamide
NH2
O
N Nicotinamide, Vitamin PP
Pyridine-3-carboxamide
Nicotinamide is known as vitamin PP, or antipellagric witamin.
Pellagra is characterized by dermatic changes (reddening, rupture,
exfoliation), gastrointestinal changes (anorexia, nausea, vomiting,
diarrhea, porphyria) and neural changes (sleeplessness, headaches,
dysmnesia, depression).
Nicotinamide is synthesised by microorganisms, among others by
intestinal flora. A precursor of nicotinamide in the body is
tryptophan.
43
The recommended daily allowance of vitamin PP is 2-12 mg for
children, 14 mg for women, 16 mg for men, and 18 mg for pregnant
or breast-feeding women. The food sources of vitamin PP are animal
products (liver, heart, kidneys, chicken, fish – tuna and salmon,
milk, eggs), fruits and vegetables (leaf vegetables, broccoli,
tomatoes, carrots, dates, sweet potatoes, asparagus, avocados), seeds
(nuts, whole grain products, legumes, saltbush seeds) and fungi
(mushrooms, brewer’s yeast). Nicotinamide in these products exists
as NAD and NADP. In therapy synthetic nicotinamide is used. The
properties of vitamin PP are also found in nicotinic acid.
Nicotinamide plays an important role in biochemical reactions. It is a
component of two coenzymes - nicotinamide-adenine dinucleotide
(NAD+) and nicotinamide-adenine dinucleotide phosphate (NADP+).
Reduced forms of these coenzymes are NADH and NADPH. The
synthesis and decomposition of NAD+ and NADP+ is shown in
Figure 58.9.
44
NICOTINAMIDEdeamidase
Nicotinate
5-Phosphoribosyl-
1-pirophosphate
PPi
Nicotinate mononucleotide (NMN)
ATP
PPi
Desamido-NAD+
ATP
AMP + PPi
Glutamine
Glutamate
NAD+ NADP+
ATP ADP
1
2
3
4
5
6
NAD+ glycohydrolase
Reduced form Oxidized form
*
N
N
O
H2
+
O
HO OH
CH2O
P
P
O
OO
O O
O
-
-N
NN
N
NH2
OHHO
O
CH2
2'
H2
N
N
Opro-R pro-S
H H
Figure 58.9.
The biosynthesis and decomposition of
NAD+ and NADP+:
– deamination of nicotinamide to
nicotinate,
– conversion of nicotinate to
nicotinate mononucleotide (NMN),
– adenylation of NMN,
– transamination (transmission of
amine group of glutamine to nicotinate
rest),
– phosphorylation of NAD+ in
position 2' of adenosyl rest,
– hydrolysis of N-glycoside bond by
NAD+ glycohydrolase.
45
Nicotinamide nucleotides act as transmitters of 2 electrons and play
an important role in red-ox reactions, catalysed by dehydrogenases. A
reactive center of the coenzyme is the position C4 of the pyridine
ring. This position is chiral. Enzymes requiring nicotinamide as an
coenzyme are stereospecific.
For example, the position C2 of ethanol is pro-chiral. Dehydrogenase
transfers stereospecifically the pro-R hydrogen atom of ethanol to the
position pro-R of NADH.
+ Alcoholic
C
CH3
H
O
N
O
NH2
R
+N
NH2
R
OHRHS
+
Ethanol
C
HS
HR
CH3
OH dehydrogenase
46
Lactate can exist as D and L, but lactate dehydrogenase of mammals
is stereospecific for L-lactate and transfers the hydrogen atom to the
position pro-R of NADH.
+
Lactate
C
CH3
COO
O
N
O
NH2
R
+N
NH2
R
OHRHS
+
-
C
COO
HO
CH3
H
-
L-Lactate
dehydrogenase
NAD+- and NADP+-dependent dehydrogenases catalyse 6 types of
reactions:
simple transmission of the hydrogen atom by alcoholic, lactate
and malate dehydrogenases
C
O
CH
OH
+ 2H+ + 2e-
47
oxidation of -hydroxy acids (isocitrate, 6-phosphogluconate
dehydrogenases)
+ CO2H2
RO
COO-
O
C
R
HCHC CH COO-
ROH
CC
oxidation of aldehydes (aldehyde dehydrogenase)
H2O+ 3 H+ + 2 e-
O-HC
O
CO
reduction of isolated double bonds
+ 2 H+ + 2 e-C CH HCC
oxidation of -CH-NH- bonds by folate reductase
+ 2 H+ + 2 e-C NH HNC
48
A deficiency of vitamin PP is observed:
in people whose main food is
maize (corn), because it contains vitamin PP in the form of
niacitin, which is not assimilated by people
sorgo, which contains a large amount of leucine which
inhibits cholinate phosphoribosyltransferase an enzyme
responsible for the conversion of tryptophan to NAD+
in people receiving isoniazid or pyrazinamide; these drugs act
antagonistically to vitamin PP (antagonistic action is also
demonstrated by pyridine-3-sulfonic acid and its amide, 3-
acetylpyridine, quinolinic acid)
in the carcinoid syndrome, in which tryptophan is transformed
to serotonine
in Hartnup disease, where the absorption of tryptophan is
disturbed.
49
58.2.8. Folic acid
NHN
N
N
N
CH2
OH
H2N1
23 4 5
678
9 10
N
OCOOH
COOH
H
Folic acid, Acidum folicum, Pteroilglutaminic acid
N-[4-[[(2-amino-4-hydroxypteridin-6-yl)methyl]-amino]benzoyl]-L-glutaminic acid
A molecule of folic acid consists of pteroic acid (comprised of 2-
amino-4-hydroxy-6-methylpteridine and 4-aminobenzoic acid) bound
with L(+)-glutaminic acid by an amide bond. The introduction of the
amine group or another amino acid into position 4 instead of
glutaminic acid produces compounds with antagonistic activity.
50
Natural folic acid, synthesized by plants and microorganisms,
contains most often from 2 to 7 glutaminic acid rests, connected by
peptide bonds in position .
In mammals monoglutaminate dominates.
Pteroylglutaminates are active and in the body are transformed into
pteroylmonoglutamate. Sources of folic acid are liver, spinach, wheat
germ, turnip greens, dried beans and peas, fortified cereal products,
sunflower seeds and other fruits and vegetables. Mammals cannot
synthesise PABA and connect pteroic acid with glutaminic acid, so
they must receive folic acid in food.
Folic acid is reduced to dihydrofolic acid by folate reductase and then
dihydrofolic acid is reduced to tetrahydrofolic acid (THF) by
dihydrofolate reductase in the presence of ascorbic acid.
51
TETRAHYDROFOLIC ACID (THF)
DIHYDROFOLIC ACID (DHF)
FOLIC ACID
H or R H or R
COOHNN
N
N
N
CH2
OH
H2N
5
9
10N
O
COOHH
DHF reductase inhibitors
e.g. methotrexate
The active forms of THF are:
N5-formyl-THF
N10-formyl-THF
N5, N10-methenyl-THF
N5, N10-methylene-THF
N5-methyl-THF
N5-formyl-THF is known as folinic acid, while N10-formyl-THF as
‘active formic acid’.
52N5-Methyl-THF
HCH3
H
5
10N
N
N
NADH + H+
NAD+
N5,N10-Methenyl-THF
N
N
N
H
5
10
NADPH + H+
NADP+
Methionine
Thymidine
Purines-C8
Purines-C2
Serine + THF
HCHO + THFN
N
N
H
H
O H
5
10
H2O
N5,N10-Methylene-THF
N
N
N
H
5
10
Histidine + THF
N10-Formyl-THF
N5-Formyl-THF
Formylmethionine10
5
HO H
H
N
N
N Methylene tetrahydrofolate is
formed from THF by the addition
of methylene groups existing in one
of three carbon donors:
formaldehyde, serine or glycine.
Methyl THF can be obtained from
methylene THF by reduction of the
methylene group.
Formyl THF is synthesised by
oxidation of methylene THF.
53
5-Methyl-THF participates together with methylcobalamin in the
conversion of homocysteine to methionine.
N5,N10-Methenyl-THF and N5-formyl-THF are donors of
monocarbon fragments in the biosynthesis of purines (the C8 and C2
atoms respectively).
N5-Formyl-THF and N10-formyl-THF are donors of the –CHO group
in the biosynthesis of formylmethionine, which makes possible the
beginning of the initiation stage in the biosynthesis of proteins.
N5,N10-methenyl-THF is the donor of the methyl group in the
biosynthesis of dTMP (deoxythymidine monophosphate) from dUMP
(deoxyuridine monophosphate)
54
The recommended daily allowance of folic acid is 25100 g for
children (depending on age), 150200 g for young and adult
people. A greater amount is required for pregnant (400 g per day)
and breast-feeding (280 g per day) women. A deficiency of folate
causes megaloblastic anemia.
Folic acid for pharmaceutical products is obtained synthetically.
The absorption of folic acid is a result of active transport and
partially of passive diffusion (2030%). In the portal circulation
THF is found. In the liver the methylation of folate is observed. The
level of folate in plasma (mainly 5-methyl-THF but also THF and
10-formyl-THF) is 717 ng/ml. In erythrocytes polyglutamates of
folate are present. The concentration of folate in erythrocytes is 40
times greater than in plasma.
5-Methyl-THF crosses the blood-brain barrier and its concentration
in the cerebrospinal fluid is 23 times greater than in plasma.
55
58.2.9 Ascorbic acid
Ascorbic acid (Acidum ascorbicum, VITAMINUM C) can be
considered as:
a furan derivative: 5(R)-5-[(S)-1,2-
dihydroxyethyl]-3,4-dihydroxy-(5H)-
furan-2-oneH
**
54 3
21
HO OH
OO
HO
H
OH
the enolic form of -lactone of 3-
oxo-L-gulonic acid H
*
*
5
4
3 21
HO OH
OO
HO
H
OH
H
*
*
5
4
3 21
OH
OO
HO
H
OH
O
56
To discuss the relationship between the chemical structure and
activity of ascorbic acid the numeration of carbon atoms is based on
that used in gulonic acid.
The endiol group is responsible for the acidic and red-ox properties
of ascorbic acid. More acidic properties are shown by the hydroxyl
group at the C3 atom (pKa=4.17) than at the C2 atom (pKa=11.57),
because the C=O group stabilizes the adjacent =C-OH group.
H
OH
OO
R
O.-O
Dehydroascorbic acid
Radical of monodehydroascorbic acidAscorbate
- e, - H++ e, + H+
OH
H
HO
OO
OH
H H
O
.O
RO
HO
H
O
O
RO
O
The endiol group of
ascorbic acid is sensitive to
oxidation.
57
As a result of the loss of one hydrogen atom by ascorbic acid a
monodehydroascorbic acid radical, stabilized by mesomerism, is
formed. The loss of another hydrogen atom results in the creation of
dehydroascorbic acid. Monodehydroascorbic radicals can also
undergo spontaneous dismutation to ascorbic acid and
dehydroascorbic acid.
The C4 and C5 carbon atoms are asymetric, so 4 isomers are
possibile L- and D-ascorbic acids and L- and D-isoascorbic acids.
There is a relationship between antiscorbutic action and the
configuration of the chiral carbon atoms. Apart from the endiol group
the R configuration at the C4 atom is necessary. This configuration is
demonstrated by natural L(+)-ascorbic acid and D(+)-isoascorbic
acid (araboascorbic acid), which shows 20 times weaker action than
L(+)-ascorbic acid. Potency is strongly increased by the S
configuration at the C5 atom. D-Ascorbic acid and L-isoascorbic acid
with the S configuration at the C4 atom do not show antiscorbutic
action.
58
When more carbon atoms or a carbinol group are introduced into the
chain activity decreases but does not disappear.
The replacement of the hydroxyl group at the C6 atom by a chlorine
atom decreases antiscorbutic action but to a lesser degree than other
changes D-Isoascorbic and L-glucoascorbic acids act
antagonistically.
The daily requirement of vitamin C is the greatest of all vitamins and
is, on average, 1 mg/kg of body mass. A greater requirement occurs
in breast-feeding women (100 mg daily).
Sources of vitamin C are fresh fruits and vegetables, especially
currants, strawberries, citruses, cabbage, parsley, spinach,
cuckooflower, tomatoes and green peppers. Foods such as eggs,
meat, bean, corn, grain products do not contain any vitamin C.
59
Primates and guinea pigs do not synthesize vitamin C because in
their bodies L-gulonolactone oxidase, which catalyses the
metabolism of L-gulonolactone to 2-oxo-L-gulonolactone, does not
exist. Other mammals can synthesize vitamin C.
D-Glucuronic acid
D-Glucose
ASCORBIC ACID
L-Gulono--lactone
L-Gulonolactone
HO
H
OH
O
OH
OH
H
O
2-Oxo-L-gulono--lactone
HO
H
O
OH
OH
H
O
O
oxidase
Figure 58.12.
The role of L-gulonolactone
oxidase in the biosynthesis of
ascorbic acid
60
7080% of vitamin C received with food is absorbed mainly in the
duodenum and in the proximal segment of the small intestine. The
active transport of vitamin C is disturbed in intestinal dysfunction,
vomiting, anorexia, alcoholism and in smokers. 25 % of vitamin C is
bound with plasma proteins.
The concentration of vitamin C in thrombocytes and lymphocytes is
higher than in erythrocytes and plasma. ASA and other salicylates,
when regularly administered, block the absorption of vitamin C by
thrombocytes and decrease its concentration in plasma and
thrombocytes.
Vitamin C is transported through the portal vein to the liver and other
tissues. The greatest amount of vitamin C is absorbed by organs with
high metabolic activity such as the adrenal glands, hypophysis,
pancreas, thymus, retina, spleen, stomach and lungs.
61
In the body, vitamin C is
oxidized to dehydroascorbate.
It is a reversible reaction and
vitamin C is regenerated
partially under the influence of
glutathione.
Additionally, dehydroascorbic
acid is metabolized to 2,3-
diketo-1-gulonic acid
(reversible reaction), which is
next metabolized to L-treonic
acid, oxalic acid, L-xylonic
acid and L-xylose (Fig.
58.13).
ASCORBIC ACID
Dehydroascorbic acid
C
C
C
C
C
COOH
H2OH
H
HO
O
O
H
OH
COOH
COOH
+
2,3-Diketo-1-gulonic acid
Oxalic acid
-CO2
+ H2O- CO2
H
H
OH
HO
H
H
CH2OH
HO
O
L-Xylose
CH2OH
COOH
H
H
HO
OH
L-Treonic acid
L-Xylonic acid
+
COOH
H
OH
HO
H
H
CH2OH
HO
L-Lixonic acid
OHH
OH
HO
H
H
CH2OH
COOH
62
The red-ox system ascorbic acid dehydroascorbic acid plays the role
of a specific hydrogen donor and a transmitter of electrons in the cells.
It forms a common red-ox system with cytochromes a and c,
pyrimidine and flavin nucleotides and with glutathione.
These properties are responsible for the participation of vitamin C in
microsomal reactions of hydroxylation catalysed by oxidases, the
control of the mitochondrial and microsomal respiratory cycle, the
control of oxidative potential in cells, the biosynthesis of folic acid,
maintaining the active forms of iron and copper [Cu(II) and Fe(II)] and
in antioxidative reactions (a scavanger of free radicals).
Additionally, vitamin C facilitates the absorption of Ca2+ ions,
stimulates the synthesis of prostaglandins and is a modulator of
immunity.
63
The following hydroxylases are ascorbic acid-dependent oxygenases:
dopamine -hydroxylase, prolyl and lysyl hydroxylase, steroid 7-
hydroxylase, 4-hydroxyphenylpyruvate dioxygenase (metaloprotein
containing Cu(II) ions) and homogentisate dioxygenase
(metaloprotein containing Fe(II) ions).
Dopamine -hydroxylase participates in the biotransformation of DA
to NA (Ch. 5.2.3.1).
The steroid 7-hydroxylase catalyses the biotransformation of
cholesterol to 7-hydroxycholesterol in the biosynthesis of bile
acids.
7-HydroxycholesterolCholesterol
7-Hydroxylase
Ascorbate
+ O2
NADPNADPH+
7 7OH
64
Prolyl and lysyl hydroxylases are peptide hydoxylases, because
hydroxylation is possibile only after the binding of proline/lysine
with polypeptide. These hydroxylases require the presence of
molecular oxygen, Fe(II) ions, -ketoglutarate as a co-substrate and
ascorbate.
Ascorbate
Fe(II)O2
OH
ProPro
Succinate-Ketoglutarate
During this reaction one oxygen atom is attached to the proline
molecule and one to the succinate molecule. Hydroxyproline and
hydroxylysine play an important role in the biosynthesis of
collagen.
65
-Ketoglutarate
Ascorbate
THYROZINE
4-Hydroxyphenylpyruvate
Glutamate
B6
Ascorbate
Homogentisate
Maleylacetoacetate
[O]
[O]
CO2
3
1
2
Thyrozine
4-Hydroxyphenylopyruvate
Homogentisate
1,2-dioxygenase
dioxygenase
aminotransferase
4-Hydroxyphenylpyruvate and
homogentisate dioxygenases
participate in the catabolism of
tyrosine.
Disturbances of tyrosine
catabolism cause such catabolic
disorders as:
type II tyrosinemia,
neonatal tyrosinemia and
alcaptonuria (congenital
metabolic disorder) (Fig. 58.14). Figure 58.14.
The participation of ascorbic acid in
the catabolism of tyrosine.
66
By reducing Fe(III) ions to Fe(II) ions ascorbic acid increases the
absorption of iron which is necessary for the production of
hemoglobin and erythrocytes. Because of that ascorbic acid is helpful
in the treatment of anemia caused by iron deficiency.
The red-ox potential of the reaction ascorbic acid dehydroascorbic
acid is E'0 = +0.1 V, which makes possible the reduction of reactive
oxygen forms: singlet oxygen (1O2), superoxide anion radical,
hydroxyl radical and other radicals. It is active in the first, second
and third defense line.
The antioxidative action of vitamin C is used increasingly in the
prophylaxis and therapy of many disorders in which overproduction
of free radicals is observed. These disorders include:
neurodegenerative disorders (Parkinson’s disease, Alzheimer’s
disease, multiple sclerosis), inflammatory diseases of the gastric tract
(Crohn’s disease, ulcerative inflammation of the colon), circulatory
system diseases (angina pectoris), neoplastic disease, rheumatism and
many others.
67
Vitamin E is the first defense line against free radicals.
As a result of its reaction with superoxide radical hydrosuperoxide
and a vitamin E radical are formed.
The vitamin E radical is reduced by ascorbic acid.
This reaction is probably mediated by coenzym Q.
The so formed monodehydroascorbic acid radical is scavanged in the
reaction of dismutation.
68
ROO
ROOHO
RX
RXH
OHOH
OH
O OH
O
L-Ascorbate
OH
OH
OH
O OH
O
_
Radical of monodehydro-ascorbic acid
L-ascorbic acid
Reaction of
OHOH
OH
OH
O
Dehydroascorbic acid
OH
OH
OHO
O O
O
HO
O
-
HO
dismutation
Figure 58.15.
The role of ascorbic acid and -tocopherol in the defense of cell
membranes against free radicals
69
It is thought that ascorbic acid inhibits the initiation and promotion
phases of neoplastic cells. (Fig. 58.16).
Ascorbic acid
Multiplication of neoplastic cells
Neoplastic cell
Normal cell
Tumor
Initiation
Promotion Ascorbic acid
Figure 58.16.
The inhibition of tumor formation
by ascorbic acid.
70
Possible anticancerogenic mechanisms of action demonstrated by
ascorbic acid:
antioxidative action
inhibition of the formation of nitrosoamines
stimulation of the immunologic system
modulation of the cancerogenic effect
protection from chromosome duplication caused by cancerogens
inhibition of the synthesis of DNA, RNA and proteins in
neoplastic cells.
71
The beneficial action of ascorbic acid has been observed in the
treatment of neoplasms of the mouth, pharynx, esophagus, stomach,
breast, lung, colon and uterine cervix.
In the physiological state, a high concentration of ascorbic acid is
observed in the stomach wall, on the luminal side. In people with
chronic gastritis, infection caused by Helicobacter pylori or
neoplasm of the stomach, low concentrations of vitamin C in plasma
and in the stomach are often observed.
Infection caused by H. pylori stimulates the formation of chronic
atrophic gastritis and leads to insufficient production of gastric acid
and a significant decrease in the level of vitamin C in the stomach.
The deficiency of gastric acid facilitates an accumulation of bacteria
which reduce nitrates to nitrites.
72
Nitrites in reactions with amines
and N-substituted amides form
nitrosamines.
Nitrosamines are very cancerogenic
for the pharynx and especially for
the stomach.
Vitamin C prevents the formation of
nitrosamines and because of that
can be helpful in the protection
against H. pylori infections and can
reduce the risk of neoplasms of the
pharynx and the stomach (Fig.
58.17).
Helicobacter pylori
Nitrosoamine
Ascorbic acid
Not damaged gastric mucosa
Atrophic gastritis
pH >5
Number of microorganisms >107/l
Nitrate Nitrite
Gastric tumor
In neoplasic diseases some specialists recommend supplementing
vitamin C by administering daily doses as high as 1 to 5 g.
73
The deficiency of ascorbic acid is accompanied by susceptibility to
infections, mucous bleeding, subcutaneous hemorrhages, edema,
arthralgia and difficult healing of wounds and fractures.
A long-term deficiency of vitamin C leads to anemia and scurvy.
When vitamin C is used in high doses, the following effects are
observed:
decreased absorption of copper because of the inhibition of the activity of
ceruloplasmin
deactivation of superoxide dismutase
release of iron from its reserves in tissues
acidification of urine, which can cause the formation of urate, citrate and
oxalate calculus in the urinary tract
disturbances of the gastric tract and diuresis
hemolysis of erythrocytes may occurs in the cases of deficiency of glucoso-
6-phosphate dehydrogenase.
74
In pharmaceutical products ascorbic acid obtained synthetically is
used.
Ascorbic acid in the solid phase and in pharmaceutical products is
stable, whereas in solutions it is rapidly degraded. In the presence of
atmospheric oxygen autooxidation to dehydroascorbic acid is
observed. This acid is hydrolysed to 2,3-diketogulonic acid which is
oxidized to oxalic acid. The products of autooxidation of ascorbic
acid are the same as its main metabolites (Fig. 58.13).
Anaerobic degradation is the effect of dehydratation, hydrolysis and
decarboxylation. The final product of these reactions is furfural (Fig.
58.18). The degradation of ascorbic acid is catalysed by metal ions.
The rate of this reaction in aqueous solutions dependens on the
concentration of hydrogen ions, substrate charge and solvent polarity.
75
Furfural
CHOO - CO2, - H2O
O
OH
COOH
O
O
O
O
O
Ascorbic acid + H2O
- 2H2O
Hydrolysis
Decarboxylation
DehydratationO
H O
O
H
H
HO
O
H
OH
O
H O
O
H
H
HO OH
O
H
Figure 58.18.
The anaerobic degradation of ascorbic acid
