VITAMINS AND COFACTORS

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VITAMINS AND COFACTORS

• By
• Henry Wormser, Ph.D.

PSC 3110 Biochemistry I - Fall 2002
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Reading material

• Principles of Biochemistry with a Human Focus by
  Garrett and Grisham, First Edition, 2002, pages
  453-468
• Handbook of NonPrescriptions Drugs, 11th edition,
  Chapter entitled Nutritional Products by Loyd
  V. Allen, Jr.

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Vitamins

• a group of organic compounds needed in small
  quantities in the diet for normal activity of
  tissues
• between 14 20 substances have been identified
  as vitamins
• many vitamins act as cofactors, coenzymes or
  prosthetic groups for enzymes
• most vitamins are derived from diet
• no calories are derived from vitamins

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Vitamins

• first vitamin discovered was thiamine or B1
• the term vitamin is derived from the fact that
  the substances are needed for life (vita) and
  because thiamine happened to be an amine the term
  was coined as such
• however, not all vitamins are amines or nitrogen
  containing compounds

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Vitamins

• vitamin requirements are usually expressed as
  RDAs (recommended dietary allowances)
• guidelines are provided by 2 organizations
• the Food and Nutrition Board of the National
  Academy of Sciences- National Research Council
• the Food and Drug Administration (FDA)

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RDAs

• applications of RDAs include
• evaluating the adequacy of the national food
  supply
• establishing standards for menu planning
• establishing nutritional policy for public
  institutions/organizations and hospitals
• evaluating diets in food consumption studies
• establishing labeling regulations
• setting guidelines for food product formulation
• developing materials for nutritional education

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RDAs

• RDAs have limitations
• they are too complex for direct consumer use
• they do not state ideal or optimal levels of
  intake
• the allowances for some categories are based on
  limited data
• the data on some nutrients in foods is limited
• they do not evaluate nutritional status
• they do not apply to seriously ill or
  malnourished patients

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Vitamin deficiencies

• primary food deficiency
• crop failure
• food storage loss
• food preparation loss
• diminished food intake
• poverty
• anorexia
• food fadism
• chronic diseases

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Vitamin deficiencies

• diminished absorption
• absorption defect
• parasites
• malignancies
• increased requirements
• rapid growth
• increased physical activity
• pregnancy
• hyperthyroidism
• increased loss
• drug therapy
• diuresis
• lactation

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Vitamin loss

• Loss is seen mainly in storage or food
  preparation
• Vitamin A sensitive to oxygen and light
• Vitamin D usually little loss
• Vitamin E sensitive to oxidation especially when
  heated or with alkali
• Vitamin K sensitive to acids, alkali, light and
  oxidizing agents
• Vitamin C very sensitive to oxidation,
  especially when heated in contact with metals
• Vitamin B complex water solubility results in
  loss in cooking water
• Riboflavin is sensitive to light

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Vitamins

• Vitamins are typically divided into 2 groups
• The fat soluble vitamins
• A, D, E, and K
• The water soluble vitamins
• The B vitamins (B1, B2, B3, B6, B7, B12 and
  pantothenic acid)
• Ascorbic acid (vitamin C)

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Bogus vitamins

• Vitamin B4 adenine
• Vitamin B10 identical with folic acid
• Vitamin B11
  
• Vitamin B15 pangamic acid
• Vitamin B13 orotic acid
• Vitamin B17 laetrile
• Vitamin B19 wormsers secret formula

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Cofactors

• provide chemical teeth for enzymes
• sometimes referred to as coenzymes
• enzymes proteins with catalytic activity
• simple enzymes large protein (polypeptide) that
  catalyzes a reaction. The enzyme gets all the
  tools (chemical teeth) it needs from the amino
  acids. However, there are only 20 different amino
  acids
• conjugated enzymes apoenzyme cofactor
  holoenzyme

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EXAMPLEProteases enzymes that cleave peptide
bonds
Enzymes perform catalytic reactions such as
hydrolysis the side chains of amino acids
participate in the reactions
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example of a simple enzyme
Usually electron-rich side chains are involved in
the catalysis Aliphatic chains are normally
involved in hydrophobic interactions
A serine protease enzyme such as chymotrypsin
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HYDROLYTIC CATALYSIS
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Example of a conjugated enzyme
Zinc protease such as ACE
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Cofactors

• all water-soluble vitamins with the exception of
  vitamin C are converted/activated to cofactors
• only vitamin K of the fat-soluble vitamins is
  converted to a cofactor
• not all vitamins are cofactors i.e., lipoic acid
  is not a vitamin
• cofactors may also act as carriers of specific
  functional groups such as methyl groups and acyl
  groups

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The water soluble vitamins
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Pantothenic acid (vitamin B5)
First recognized in 1933 as a growth factor for
yeast (Roger J. Williams)
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Pantothenic acid

• a yellow viscous oil (free acid)
• stable to moist heat (not to dry heat) and to
  oxidizing and reducing agents
• hydrolyzed in acid or alkaline medium
• sources (numerous) liver, kidney, eggs, lean
  beef, milk, molasses, cabbage, cauliflower,
  broccoli, peanuts, sweet potatoes, kale (derive
  its name from everywhere)

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Pantothenic acid

• serves in its activated form as the cofactor for
  coenzyme A (CoA) and the acyl carrier protein
  (ACP)
• first phosphorylated by ATP to 4-phosphopantothen
  ate
• next is the formation of 4-phosphopantetheine by
  addition of cysteine and decarboxylation
• adenylation by ATP forms dephospho-CoA
• phosphorylation to the 3-OH of the ribose
  generates CoA (coenzyme A)

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Coenzyme A

• performs a vital role by transporting acetyl
  groups from one substrate to another
• the key to this action is the reactive thioester
  bond in the acetyl form of CoA
• the thioester bond is stable enough that it can
  survive inside the cell, but unstable enough that
  acetyl-CoA can readily transfer the acetyl group
  to another molecule

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Example of an acetylation reaction
Acetylcholine is an important neurotransmitter
in the autonomic nervous system (cholinergic) and
in the brain
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Pantothenic acid

• Deficiency
• rats
• graying of hair/fur in black rats
• dermatitis
• inflammation of nasal mucosa
• hemorrhage of adrenal cortex
• humans
• has not been encountered or extremely rare
• difficult to induce with either synthetic diets
  and/or with antagonists (omega-methylpantothenic
  acid

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Pantothenic acid

• vague symptoms in human deficiency
• numbness and tingling in feet burning foot
• fatigue
• GIT disturbances
• available pharmaceutically as calcium
  pantothenate (d-isomer) and as racemic mixture
• 5 - 7 mg/day appear to prevent signs of
  deficiency
• appears to be non-toxic (up to 10-20 gm have been
  tolerated)

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Thiamine
Vitamin B1 antiberi-beri vitamin antineuritic
factor was the first water soluble vitamin
discovered (Eijkman)
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Thiamine

• has the odor and flavor of yeast
• slowly destroyed by moist heat more rapidly
  destroyed in a basic medium than in an acid one
• source whole cereals and grains yeast organ
  meat
• pharmaceutical products use the hydrochloride or
  mononitrate salts

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Thiamine

• active form is thiamine pyrophosphate (formed by
  the action of thiamine diphosphotransferase)
• involved in the oxidative decarboxylation of
  pyruvic acid and a-ketoglutaric acid
• involved in the transketolase reactions of the
  triose phosphate pathway
• also required for nerve function (unrelated to
  coenzyme activity)

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Conversion of thiamine to TPP
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Typical reactions catalyzed by TPP
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Reactions in which thiamine pyrophosphate is a
cofactor

• Pyruvate decarboxylase
• Alcohol fermentation pyruvate to acetaldehyde
• Pyruvate dehydrogenase
• Synthesis of acetyl-CoA
• Alpha-ketoglutarate dehydrogenase
• Citric acid cycle
• Transketolase reaction
• Carbon-fixation reactions of photosynthesis
• Acetolactase synthetase
• Valine, leucine biosynthesis

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Thiamine pyrophosphate

• the key portion of this cofactor is the
  thiazolium ring with its acidic hydrogen
• the hydrogen is removed by the enzyme forming an
  ylid (anion next to cation)
• the anion can then react with carbonyl groups in
  such molecules as pyruvate
• the pyrophosphate functionality acts as a
  chemical handle which holds the cofactor in place
  within the enzyme

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Chemical mechanism for action of B1 in
pyruvate dehydrogenase
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Transketolase reaction
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Transketolase reaction
These reactions provide a link between the
pentose phosphate pathway and glycolysis Activity
of erythrocyte transketolase is commonly used as
an index of thiamine deficiency
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Thiamine deficiency

• earliest symptoms of thiamine deficiency include
• constipation
• appetite suppression
• nausea
• mental depression
• peripheral neuropathy
• fatigue

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Thiamine deficiency (severe)

• beri-beri (once associated with white polished
  rice diets and with highly milled wheat diets)
• 2 clinical types
• dry beri beri or neuritic beriberi
• associated with polyneuropathy (depressed
  peripheral nerve function, sensory disturbance,
  loss of reflexes and motor control and muscle
  wasting
• wet beri beri or cardiovacular beriberi
• edema, congestive heart failure

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These 2 compounds are potent antithiamine agents
which may be used to induce symptoms of vitamin
B1 deficiency in selected animals. Oxythiamine
competitively inhibits thiamine pyrophosphate and
becomes active after phosphorylation
neopyrithiamine prevents the conversion of
thiamine to thiamine pyrophosphate
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Other clinical applications

• Alcohol neuritis (peripheral neuropathy)
• Sharp burning pain in the feet
• Deep muscle tenderness with numbness
• Coarse tremors, foot drop
• Wernickes encephalopathy
• Results from degeneration of basal ganglia due to
  chronic/heavy use of alcohol
• Rigidity of extremities
• Complete or partial ophthalmoplegia
• Sleep disturbances
• Nausea and vomiting

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Other clinical applications

• Korsakoffs syndrome or psychosis
• Also a complication of chronic/heavy use of
  alcohol
• Usually follows DTs (delirium tremens)
• Memory loss
• Delusions
• Disorientation
• Ocular palsies
• Combined Wenicke-Korsakoff syndrome
• Pregnancy neuritis
• Certain gastrointestinal disorders

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Requirement for thiamine

• Based on energy needs
• 0.3 0.6 mg/1000 calories
• Increased requirements
• Pregnancy and lactation
• Eating large amounts of raw sea food (clams)
  contain thiaminase
• Stress situations (high level of exercise, fever,
  hyperthyroidism)
• Drinking large quantities of tea (contains
  antagonist)

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Thiamine assay

• biologic assay in animals time consuming and
  costly (curative or protective)
• microbiologic using bacteria which require
  thiamine for growth
• chemical/fluorescent assay conversion of
  thiamine to thiochrome by alkaline ferricyanide

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Lipoic acid

• lipoic acid is a co-factor found in pyruvate
  dehydrogenase and a-ketoglutarate dehydrogenase,
  two multienzymes involved in a-keto acid
  oxidation
• lipoic acid functions to couple acyl group
  transfer and electron transfer during oxidation
  and decarboxylation of a-ketoacids
• no evidence exists of a dietary lipoic acid
  requirement in humans therefore it is not
  considered a vitamin

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Lipoic acid exists in 2 forms a closed-ring
disulfide form and an open-chain reduced form
oxidation-reduction cycles interconvert these 2
species lipoic acid exists covalently attached
in an amide linkage with lysine residues on
enzymes
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Riboflavin

• vitamin B2, lactoflavin (ovo, hepato, verdo),
  vitamin G
• a heterocyclic flavin linked to ribose analogous
  to the nucleosides in RNA
• orange-yellow fluorescent compound
• found in significant quantities in green leafy
  vegetables, milk and meats
• heat stable, but easily destroyed by light
• recommended intake is related to energy intake
  (kcal) RDA 1 2 mg/day

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dimethylisoalloxazine ring system confers
some degree of planarity to the molecule and
also color (yellow)
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Decomposition of riboflavin
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Riboflavin

• 2 cofactors are involved
• riboflavin phosphate (flavin mononucleotide, FMN)
• flavin adenine dinucleotide (FAD)
• involved in the metabolism of carbohydrates, fats
  and proteins (flavin dehydrogenases/flavoproteins)
  
• hydrogen carriers in the respiratory chain

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Riboflavin
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Riboflavin
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Riboflavin

• Enzymes utilizing riboflavin cofactors
• NADH dehydrogenase
• succinate dehydrogenase
• d and l-amino acid oxidases
• pyridoxine-5-phosphate oxidase
• glutathione reductase
• xanthine oxidase
• In some enzymes, the cofactor is covalently
  bonded to an amino acid (dehydrogenases)

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Dehydrogenase reaction
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Amino acid oxidases
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Xanthine oxidase
Xanthine oxidase is a flavoprotein which also
contains Fe and Mo
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Fatty acyl-CoA desaturase
Important step in the biosynthesis of unsaturated
fats this reaction is actually more complex than
shown here and involves other cofactors, but FAD
is a key cofactor for the enzyme
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Riboflavin deficiency

• seldom seen in industrialized societies
• deficiency when seen
• cheilosis (vertical fissure in the lips)
• angular stomatitis (craks in the corner of the
  mouth)
• glossitis
• photophobia
• seborrheic dermatitis
• normochromic normocytic anemia
• usually encountered along with pellagra (niacin
  deficiency)
• newborns treated for hyperbilirubinemia by
  phototherapy (riboflavin is unstable to light)

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Biotin
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Biotin

• an imidazole sulfur containing compound
• sometimes referred to as vitamin B7 or vitamin H
• widely distributed in foods (liver, kidney, milk,
  molasses)
• a large portion of the daily need of biotin is
  met by synthesis by intestinal bacteria
• deficiency is usually the result of a defect in
  utilization rather than simple dietary deficiency

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Biotin

• like lipoic acid, biotin is converted to its
  coenzyme form (called biotinyllysine or biocytin)
  by formation of a covalent amide bond to the
  nitrogen of a lysine residue
• like lipoic acid it performs a highly specialized
  function adds a carboxyl group to substrates

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Biotin

• biochemical role carbon dioxide fixation
• two step process
• Binding of CO2 to biotin N-carboxybiotin
• Transfer of CO2 to a substrate
• Activation of biotin requires enzyme, CO2, ATP
  and Mg

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Biotin

• Biotin-dependent enzymes
• Pyruvate carboxylase (synthesis of oxaloacetate
  for gluconeogenesis and replenishment of the
  citric acid cycle)
• Acetyl CoA carboxylase (fatty acid biosynthesis)
• Propionyl-CoA carboxylase
• b-methylcrotonyl-CoA carboxylase
• holocarboxylase synthase (multiple carboxylase)

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Reactions involving biotin enzymes
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Biotin

• deficiency
• quite uncommon
• can be induced by feeding raw egg white (avidin)
• avidin is a protein which binds tighly with
  biotin (MW 70,000)
• symptoms are anorexia, nausea, muscle pain, fine
  scaly desquamation of the skin
• requirements 150 200 mcg/day
• therapeutic use in babies with infantile
  seborrhea (cradle cap) and Leiners disease

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Pyridoxine (vitamin B6)
A pyridine derivative
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Other forms of B-6
Collectively, pyridoxine, pyridoxal and
pyridoxamine are known as vitamin B6
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Pyridoxine

• vitamin B6, rat acrodynia factor,
  antidermatitis factor
• widespread occurrence
• pyridoxine mostly in vegetable products
• pyridoxal and pyridoxamine mostly in animal
  products
• pyridoxine is stable in acid solution, but
  unstable in neutral or alkaline solutions
  (destroyed by light)

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Pyridoxal phosphate

• pyridoxine is converted to pyridoxal phophate by
  phosphorylation and oxidation to the aldehyde
• pyridoxal phosphate is then attached to the
  holoenzyme via a covalent bond to a lysine
  residue (a Schiffs base)
• the Schiffs base bond is readily broken and
  reformed
• this reversibility is very important in the
  biochemical action of this cofactor

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Biochemical functions
Able to catalyze the breakdown of amino acids
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Pyridoxal phosphate

• Biochemical functions
• Decarboxylation of amino acids
• Transaminase reactions
• Racemization reactions
• Aldol cleavage reactions
• Transulfuration reactions
• Conversion of tryptophan to niacin
• Conversion of linoleic acid into arachidonic acid
  (prostaglandin precursor)
• Formation of sphingolipids

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Decarboxylation of amino acids
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Important transaminases

• ALT ( alanine aminotransferase)
• formerly known as SGPT (serum glutamate pyruvate
  transaminase)
• alanine alpha-ketoglutarate pyruvate
  glutamate
• increased serum level in liver injury

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Important transaminases

• AST (aspartate aminotransferase)
• formerly known as SGOT (serum glutamate
  oxaloacetate transaminase)
• aspartate alpha-ketoglutarate oxaloacetate
  glutamate
• elevated when heart and/or liver are damaged

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Important decarboxylases
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Mechanism for transamination reaction
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Pyridoxine

• deficiency
• difficult to produce in humans
• may be accomplished artificially with a
  pyridoxine antagonist (deoxypyridoxine)
• symptoms include nausea and vomiting, seborrheic
  dermatitis, depression and confusion, mucous
  membrane lesions, peripheral neuritis, anemia

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Pyridoxine antagonists
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Pyridoxine can antagonize the antiparkinsonian
use of L-DOPA
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Pyridoxine deficiency

• can be monitored by measuring the level of
  xanthurenic acid in the urine
• this is related to a decrease in kynureninase
  activity (pyridoxal phosphate is the coenzyme)
• kynurenine, a breakdown product of tryptophan is
  normally converted to kynurenic acid but in B6
  deficiency it is shunted to form xanthurenic acid

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XANTHURENIC ACID
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Pyridoxine

• requirements
• children 0.5 1.2 mg
• adults 2.0 mg
• pregnancy 2.5 mg
• Requirement for B6 is proportional to the level
  of protein consumption
• therapeutic uses
• deficiency
• to counterract the effects of antagonists
• certain rare forms of anemia
• in women taking oral contraceptives (estrogen
  shifts tryptophan metabolism

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Discovered in 1913 from yeast also known as
vitamin B3
1915 1920 Irving Golberg demonstrated that
lack of niacin causes pellagra
one of the simplest vitamin like B6 also a
pyridine derivative
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Oxidation of nicotine yields nicotinic acid
This reaction does not occur in vivo strictly a
laboratory reaction
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Nicotinic acid

• niacin, vitamin B3, niacinamide, antipellagra
  vitamin
• both form are active the free acid and the amide
• sources organ meat (largest source), fish,
  yeast, dried fruit, nuts, cereal grains, some
  vegetables
• pellagra-inducing diets corn meal, corn starch,
  sweet potatoes, rice, syrup, pork fat (once a
  common diet in southern states among
  sharecroppers)

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Coenzyme forms
Two cofactor forms of niacin NAD and NADP these
cofactors are not tightly held by the enzyme and
may be reused for reaction after reaction
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Biochemical function
In the older literature NAD is referred to as
DPN or coenzyme I NADP is referred to as TPN or
coenzyme II
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Oxidized and reduced forms
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Sparing action of tryptophan
Tryptophan can substitute for niacin 60 mg of
tryptophan is equivalent to 1 mg of niacin 60 gm
of protein contains 600 mg of tryptophan which
then represent 10 mg of niacin
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Pellagra

• Early stages
• Anorexia
• Indigestion
• Muscle weakness
• Reddened skin
• Rough skin
• Advanced stages
• 3 Ds of pellagra dermatitis, diarrhea, dementia

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Clinical uses of nicotinic acid

• pellagra symptoms from
• gastric ulcer or carcinoma
• diarrhea
• isoniazid therapy
• carcinoid syndrome
• Hartnup disease (impairment of tryptophan
  absorption)
• peripheral vasodilator (nicotinic acid or
  nicotinyl alcohol)
• hypolipidemic agent (only nicotinic acid in large
  doses lowers both triglycerides and cholesterol
  (Niaspan, Nicobid)

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Carcinoid syndrome

• a slow growing neoplasm of enterochromaffin cells
  (ileum, stomach, bronchus)
• tryptophan metabolism is altered resulting in
  excess serotonin synthesis
• symptoms include
• facial flushing
• edema of head and neck
• abdomina cramps and diarrhea
• asthmatic symptoms
• cardiac insufficiency
• urinary 5-HIAA (5-hydroxyindole acetic acid) is
  high (5-HIAA is a metabolite of serotonin
  serotonin is derived from tryptophan)

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Cautions concerning the use of nicotinic acid in
large doses

• as an acid, it can erode gastrointestinal mucosa
  leading to ulceration
• it also causes a depletion of glycogen stores and
  fat reserves in skeletal and cardiac muscle
• additionally, there is an elevation in blood
  glucose and uric acid production
• for these reasons, nicotinic therapy is not
  recommended for diabetics or persons who suffer
  from gout

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Ascorbic acid

• vitamin C anti-scorbutic vitamin (scurvy)
• structure is reminiscent of glucose
• produced in plants from glucose via the uronic
  pathway
• the enzyme gulonolactone oxidase converts
  gulonolactone to ascorbic acid
• exists in the enolic and ketonic forms
• sources citrus fruits, tomatoes, green peppers,
  strawberries, cantaloupe, cabbage, turnips, peas,
  lettuce and aspargus

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ASCORBIC ACID AND DEHYDROASCOBIC ACID
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Ascorbic acid

• Biochemical functions
• Production and maintenance of collagen
• Proline --------hydroxyproline
• Lysine -------- hydroxylysine
• Mitochondrial electron-transport chain
  (cytochrome C)
• Metabolism of tyrosine
• Tyrosine ----- p-hydroxyphenylpyruvic acid----
  2,5-dihydroxyphenylacetic acid (homogentisic acid)

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Proline hydoxylase (collagen formation)
Dopamine-beta hydroxylase ( neurotransmitter
formation)
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Anti-oxidant properties of vitamin C helps
prevent damage to cellular proteins and DNA
Normal metabolic processes in the cell lead to
the generation of reactive oxidizing agents such
as superoxide Superoxide can react with and
damage protein and DNA, leading to cellular
changes that can lead to premature aging and
cancer Vitamin C reacts with superoxide, thus
preventing this damage
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Ascorbic acid

• conversion of folic acid to THFA
• hydroxylation reactions of cholesterol to cholic
  acid
• hydroxylation of tryptophan to 5-hydroxytryptophan
  
• regulation of cholesterol biosynthesis in the
  adrenal gland
• aids in the absorption and utilization of iron
• antioxidant properties may inhibit formation of
  nitrosamines during digestion of protein

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Ascorbic acid

• defiency scurvy
• hemorrhage from mucous membranes, mouth and GIT,
  skin and muscles
• gingivitis swelling, tenderness, redness and
  ulceration of gums
• loosening or loss of teeth
• swelling of joints
• rarefaction of bones and dentine

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Ascorbic acid

• requirements
• children 30 mg
• adults 40 80 mg
• pregnancy 100 mg
• therapeutic uses
• scurvy
• idiopathic methemoglobinemia
• questionable use common cold

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Vitamin B12
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Vitamin B12

• cyanocobalamin (Redisol)
• hydroxocobalamin (Alpha redisol)
• function
• deficiency
• hematological sequelae
• neurological sequelae

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Vitamin B12

• synthesized by bacteria only
• red in color, levorotatory and stable to heat
• commercially available either as cyano or
  hydroxocobalamin
• stored in the liver as the coenzyme
• absorbed only in the presence of the intrinsic
  factor (a glycoprotein released by parietal
  cells)
• transported to tissues via transcobalamin II
• present in foods such as liver, fish, eggs, milk
• absent in vegetables and fruits

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Vitamin B12

• by far the most complex vitamin in structure
• made up of a planar corrin ring (4 pyrroles)
• the only vitamin that possesses a metal ion
  (cobalt) as part of its structure
• the major cofactor form of B12 is
  adenosylcobalamin or 5-deoxyadenosylcobalamin
• small amounts of methylcobalamin also occur
  (intermediate in methyl transfer reactions)

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Vitamin B12

• the corrin ring is similar to the porphyrin ring
  system found in hemoglobin except that in corrin
  2 of the pyrroles are linked directly (without
  methylene bridges)
• the cobalt is coordinated to the 4 pyrrole
  nitrogens
• one of the axial cobalt ligands is a nitrogen of
  the dimethylbenzimidazole group
• the other axial ligand may be CN, OH, CH3 or the
  5-carbon of a 5-deoxyadenosyl group

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corin nucleus
cobalt coordinated
benzylimidazole
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Vitamin B12

• biochemical functions (mediated by coenzymes)
• mutase reaction (rearrangement reaction
• methylmalonyl CoA to succinyl CoA (lipid
  metabolism)
• methylation reactions
• uracil to thymine
• homocysteine to methionine
• aminoethanol to choline
• activation of amino acids for protein synthesis
• ribonucleotides to deoxyribonucleotides for DNA
  synthesis in certain bacteria

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Causes of B12 deficiency

• Pernicious anemia (autoimmune gastritis against
  parietal cells - loss of intrinsic factor)
• rarely due dietary deficiency
• N2O/oral contaceptive drugs
• intestinal parasite
• gastrectomy
• chronic gastritis
• Schilling test

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Diagnosis of B12 deficiency

• Schilling test
• distinguishes deficiency caused by pernicious
  anemia with that caused by malabsorption
• compares absorption in radiolabeled B12 with
  intrinsic factor and radiolabeled B12 without
  intrinsic factor
• in pernicious anemia the B12 with intrinsic
  factor will be absorbed while the B12 by itself
  will not
• in malabsorption neither will be absorbed

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Manifestation of B12 deficiency

• macrocytic megaloblastic anemia
• megaloblasts are abnormal erythroid precursors in
  bone marrow (most cells die in the bone marrow)
• reticulocyte index is low
• hyperchromic macrocytes appear in blood
• anemia reflects impaired DNA synthesis
• other cells may be involved (leukopenia,
  thrombocytopenia
• spinal cord degeneration (irreversible)
• swelling, demyelination, cell death
• neurological disease
• results from deficient methylmalonyl-CoA mutase
• this cannot be treated with folic acid!!

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Treatment of B12 deficiency

• use IM cyanocobalamin or hydroxocobalamin
• administer daily for 2 - 3 weeks, then every 2 -
  4 weeks for life
• monitor reticulocytosis early to assure treatment
  is working (reticulocyte count should go up)
• monitor potassium levels to ensure hypokalemia
  does not occur due to excessive RBC synthesis

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Folic acid

• MOA
• deficiency
• use
• drug interactions with folic acid

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Also known as folacin, vitamin M and
pteroylglutamic acid Widely distributed in leaves
(foliage) of plants
Chemically composed of pteroic acid (pteridine
and PABA) and glutamic acid
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FOLIC ACID

• absorbed by both active and passive transport
• on the average we absorb 50 -200ug per day (about
  10 -25 of dietary intake)
• storage is in the form of 5-methyl THF (5 -20
  mg)
• found in green vegetable, dietary yeasts, liver,
  kidney
• bacteria synthesize their own folic acid
  (dihydropteroate synthetase)

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Folic acid

• Biochemical functions
• one carbon fragment transfer (formyl, methyl,
  hydroxymethyl)
• conversion of homocysteine to methionine
• conversion of serine to glycine
• synthesis of thymidylic acid
• synthesis of purines (de novo)
• histdine metabolism
• synthesis of glycine

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PURINE CARBONS DERIVED via FOLATE
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Deficiency of folic acid

• Inadequate intake
• defective absorption (most common)
• sprue
• gastric resection and intestinal disorders
• acute and chronic alcoholism
• drugs (anticonvulsants and oral contraceptives)
• pregnancy
• pellagra

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Deficiency of folic acid

• abnormal metabolism of folates
• folic acid antagonists (dihydrofolate reductase
  inhibibitors - methotrexate, pyrimethamine,
  trimethoprim)
• enzyme deficiency
• vitamin B12 deficiency
• oral contraceptives
• increased requirement
• pregnancy, infancy

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METHOTREXATE
Inhibits enzyme dihydrofolate reductase (DHFR)
which is necessary for maintaining pool of
reduced folates required for DNA synthesis
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METHOTREXATE

• also known as amethopterin or MTX
• a potent inhibitor of dihydrofolate reductase
  which catalyzes the conversion of folic acid to
  tetrahydrofolic acid (THFA)
• THFA acts as an acceptor of a one-carbon unit
  from either formate or formaldehyde
• 5-formyl THFA is also known as folinic acid or
  the citrovorum factor (leucovorin)
• THFA one-carbon carriers are important in the
  synthesis of purines, thymine, choline, and other
  important cellular constituents
• MTX is used in treating acute lymphocytic
  leukemia in children, choriocarcinoma, osteogenic
  sarcoma, carcinomas of the head, neck, bladder
  and testis
• in lower doses treatment of psoriasis and
  rheumatoid arthritis

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• diaminopyrimidines inhibitors of dihydrofolate
  reductase
• have activity in both bacterial and protozoal
  organisms
• more effective if used in combination with
  another drug
• pyrimethamine is more selective for protozoal
  enzyme than trimethoprim
• used in treatment of malaria and PCP

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The fat soluble vitamins

• By
• Henry Wormser
• Professor of Medicinal Chemistry

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Fat soluble vitamins

• Vitamins A, D, K and E are the fat-soluble
  vitamins
• excessive use of vitamins A and K can lead to
  toxicities
• fat soluble vitamin tend to be stored in fatty
  tissues of the body and in the liver

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Vitamin A

• Exits in 3 forms
• all trans-retinol
• long chain fatty acyl ester of retinol (main
  storage form)
• retinal (the active form in the retina)
• retinoic acid is also considered to be
  physiologically active
• provitamin A or carotene can be converted to
  retinol in vivo

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Vitamin A

• recommended intakes are expressed in retinol
  equivalents (RE)
• 1 RE 1 mcg of retinol
• 6 mcg of b-carorene
• 12 mcg other carotenes
• older usage expressed activity in USP units or
  International units (IU). These were based on
  biological activity in the vitamin a-deficient
  rat (1 IU 0.3 mcg of retinol)

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Vitamin A contains 5 conjugated double bonds
which are key to some biological actions Isolated
in impure form by McCollum in 1915
RDA 0.7 mg
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Vitamin A

• Diseases of deficiency
• Nigh blindness and xerophthalmia (dry eye)
• Skin disorders
• Lack of growth
• Hypervitaminosis
• A serious potential problem (CNS disorders birth
  defects)

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Vision and the role of vitamin A

• photoreception is the function of 2 specialized
  cell types rods and cones
• both types of cells contain a photosensitive
  compound called opsin
• in rod cells opsin is called scotopsin and the
  receptor is called rhodopsin or visual purple
• rhodopsin is a serpentine receptor imbedded in
  the membrane of the rod cell it is a complex
  between scotopsin and 11-cis retinal

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Vision and the role of vitamin A

• intracellularly, rhodopsin is coupled to a
  G-protein called transducin
• when rhodopsin is exposed to light, it is
  bleached releasing the 11-cis-retinal from opsin
• absorption of photons by 11-cis-retinal triggers
  the conversion to all-trans-retinal (one
  important conformational intermediate is
  metarhodopsin II) also there is a change in
  conformation of the photoreceptor

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Vision and the role of vitamin A

• these transformations activate a
  phosphodiesterase (which hydrolyzes c-GMP to GMP)
• c-GMP is necessary to maintain the Na channels
  in the rods in the open conformation
• with a decrease in c-GMP, there occurs a closure
  of the Na channels, which leads to
  hyperpolarization of the rod cells with
  concomittant propagation of nerve impulses to the
  brain

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Additional role of retinol

• retinol also functions in the synthesis of
  certain glycoproteins and mucopolysaccharides
  necessary for mucous production and normal growth
  regulation
• this is accomplished by phosphorylation of
  retinol to retinyl phosphate which then functions
  similarly to dolichol phosphate

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Retinoic acid (Retin-A) is important for cellular
differentiation It controls cellular growth
particularly cell growth Used in the treatment of
acne also used as an anti-wrinkle agent (Retin
A, Retin A micro, Avita, Renova)
Also used orally to treat acute promyelocytic
leukemia (APL) Product used is Vesanoid (10 mg
capsules)
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Isotretinoin or accutane is a modification of
retinoic acid it contains a 13-cis double bond
and is orally effective Used in the treatment of
severe acne
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An aromatic analog of retinoic acid orally
effective and used in the management and
treatment of psoriasis
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Etretinate (Tegison)
Esterified form of acitretin also used orally in
the treatment of recalcitrant psoriasis 10 and
25 mg capsules
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Alitretinoin (Panretin)
Currently used as a 0.1 gel for the topical
treatment of cutaneous lesions in patients with
AIDS-related Kaposi sarcoma
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BEXAROTENE (Targretin)
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Bexarotene (Targretin)

• indicated for the treatment of cutaneous
  manifestations of cutaneous T-cell lymphoma
• usually the patients receiving this drug have
  failed to respond to other treatment protocols
• pregnancy (Category X drug)

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Adapalene (Differin)
Used as a 0.1 gel in the treatment of acne
vulgaris
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Tazarotene (Tazorac)
Topical treatment of patient with facial acne
vulgaris of mild to moderate severity gel
0.05, 0.1
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Vitamin A toxicity

• vitamin A is higly toxic when taken in large
  amounts either acutely or chronically
• may occur with 200 mg (666,000 IU) in adults or
  half this amount in children
• signs include headache, nausea and vomiting,
  increased cerebrospinal fluid pressure, blurred
  vision and bulging of the fontanelle in infants

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Vitamin D

• There are 2 major precursor forms
• 7-dehydrocholesterol
• ergosterol
• UV irradiation affords cholecalciferol (vitamin
  D3) and ergocalciferol (vitamin D2)
• Discovery
• 1890 sunlight prevents rickets
• 1924 Steanbock and Hess found that irradiating
  certain foods produced vitamin D2
• 1970 hormonally active form of vitamin D
  discovered

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Vitamin D

• RDA 20 mg (required in minute amounts)
• disease of deficiency rickets
• Malformation of bones due to improper bone
  mineralization
• Hypervitaminosis
• Toxic dose only 10X higher than the RDA
• Causes hypercalcemia can lead to cardiac arrest
• vitamin D is not a vitamin (or a cofactor) it
  is a steroid hormone

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Biological functions

• Calcium homeostasis it is critical for the body
  to maintain the proper calcium level in the blood
  stream
• Intestinal calcium absorption acts as a signal
  to tell intestinal cells to take up more calcium
  from the gut
• Bone calcium mobilization
• Signals osteoclast (bone cells) to release
  calcium into the blood stream in response to low
  calcium levels

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Biological functions

• Cellular differentiation much less well
  understood signal to bone marrow cells to
  change into other cells

Problem 1a,25(OH)2-D3 causes hypercalcemia
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Various analogs of vitamin D
Potential use -anti-cancer agent
-immunosuppressive
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Doxercalciferol (Hectorol)

• a synthetic vitamin D analog that undergoes in
  vivo metabolic activation to 1-a,25-dihydroxyvitam
  in D2
• Activation does not require involvement of the
  kidneys
• Used in hyperparathyroidism in patients
  undergoing chronic renal dialysis
• Initial dose 10 mcg orally 3 times per week

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PARICALCITOL (Zemplar)
A synthetic vitamin D analog indicated for the
prevention and treatment of secondary hyperparathy
roidism associated with chronic renal failure
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Calcipotriol (Dovonex)
a vitamin D derivative approved for the
treatment of psoriasis. Mechanism of action is
unknown. Receptor affinity is similar to that of
calcitriol, but is less than 1 as active in
regulating calcium metabolism
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Calcipotriene

• An analog of vitamin D3 with a modified
  side-chain containing a 24-OH group and a
  cyclopropyl group
• binds strongly to the D3 receptor on
  keratinocytes in skin and it suppresses their
  proliferation (used in psoriasis)
• has only about 0.5 of the activity of D3 on
  calcium and phosphorus metabolism

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Dihydrotachysterol (DHT)
A reduction product of vitamin D-2 Used in the
management of hypoparathyroidism has only 1/450th
the antirachidic activity of vitamin D-2
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Vitamin K

• the koagulation vitamin
• exists in 2 forms
• plant origin phylloquinone or vit K1
• bacterial origin menaquinones or vit K2
• also certain synthetic quinones have vitamin K
  activity
• menadione (vitamin K3)
• menadiol sodium phosphate (vitamin K4)

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Vitamin E

• alpha (E1), beta (E2) and gamma(E3) tocopherol
• sources plant oils (corn, peanut, wheat germ),
  green leafy vegetables, meat, eggs
• value resides in the antioxidant properties of
  vitamin E (may prevent the formation of
  peroxides)

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ALPHA TOCOPHEROL
Found in a variey of different sources (primarily
vegetable fats)
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Vitamin E

• Estimated requirements 5 mg/day 0.6 mg/day of
  unstaurated fat
• Biological function antioxidant for fatty acids
• Acts like vitamin C prevents lipid peroxidation
  and/or damage to cells by lipid hydroperoxides

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Uses for vitamin E

• hemolytic anemia in premature infants,
  unresponsive to B12, Fe and folic acid
• macrocytic megaloblastic anemia seen in children
  with severe protein-calorie malnutrition

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Other coenzymes
Serves as entry into the electron- transport chain

Involved in the conversion of phenylalanine to
tyrosine
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The End
09/12/02