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Learning Objectives
• Recognize the structures of vitamins • Describe the main biochemical and biological functions of each vitamin • Outline and classify main metabolic reactions that each vitamin participates
in • Identify the symptoms and common disease name related to a particular
vitamin deficiency • Apply chemical or biochemical methods to diagnose deficiency of a
particular vitamin • Evaluate the risk factors and/or populations for each vitamin deficiency • Describe and apply ways to treat a particular vitamin deficiency • Recommend the use of certain vitamin on relevant diseases or conditions • Recognize and explain interactions of certain drugs with vitamins • Identify the toxicities of vitamins when overdosed (rare in water-soluble
vitamins, but common in fat-soluble ones) • Explain the interactions between some vitamins
At the end of the course, you should be able to:
Related to Their High Water Solubility
• Metabolism and storage -- only B12 and folate (B9) are appreciably stored. In general, water soluble vitamins are excreted readily and are not stored. As a result, depletion is more of a problem than toxicity.
• Toxicity -- only niacin (b3) and pyridoxine (b6) are at all toxic (in high conc.). In general, the water soluble vitamins have few toxicities.
Common Functions • The water soluble vitamins are coenzymes for
various common biochemical reactions • Their status can be readily determined by
measuring the appropriate enzyme activities in blood. – Typically, the enzyme activity is measured in the
absence and the presence of exogenously added coenzyme, to determine whether the patient needs more of the vitamin.
OHSN+
N
N
Thiamin
TMP TPP TTPATP ATP ATP
NH2
H
Thiamin (B1) - Structure
Pyrimidine ring
Thiazole ring
pKa = 17 - 19
Absorption and Transport
• Active transport. Occurs at low concentrations. Two thiamin transporter has been identified in intestine: ThTr1 and ThTr2. After cellular uptake, thiamin is converted to TPP. Mutations in ThTr1 gene is associated with thamin-responsive megaloblastic anemia (TRMA).
• Passive diffusion. At high concentration (2.5 mg dose for a human), passive diffusion also occurs.
• Serum. Most thiamin is serum is bound to protein, mainly albumin, non-specifically. About 90% of total thiamin in blood is contained in erythrocytes.
Metabolism
• Phosphorylation: TMP, TPP, TTP • Catabolism: TTP and TPP are catabolized by
thiamin pyrophosphatase to give TMP. Thiamin has an estimated half-life of 10-20 days in human so thiamin deficiency states can deplete tissue stores within a couple of weeks.
• Excretion: Excreted in urine, mainly as free thiamin and TMP, along with over 20 metabolites.
Function – Cofactor for Enzymes Used to Harvest Carbohydrates for Energy
Enzyme cofactor - Oxidative decarboxylation of a-keto acids
+
CH3 C CO2HO
TPP
lipoic acidNAD
CoASHpyruvate dehydrogenase complex
CH3 C SCoAO
CO2e.g. + + NADH
e.g. α -ketoglutaric acid TPP succinyl CoA CO2
NADCoASH
++
lipoic acidα-ketoglutarate dehydrogenase complex
The decarboxylation is accomplished by a mitochondrial enzyme complex as shown in next slide.
Pyruvate dehydrogenase complex
E LSH
SH
CoASH
CH3 CSCoAO
NAD+
TCAcycle
E TPP
E TPP
C OH
CH3
(α-hydroxyethyl TPP)
E LS
S
E L SH
S C CH3O
CH3 C COOHO
CO2
NADH
L = lipoic acid, E = enzyme, TPP = thiamin pyrophosphate.
Transfer of a-ketols (pentose phosphate pathway)
CHOHCHO
CH2OPO3H2
+O=
HOCHCCH2OH
HCOH
HCOH
HCOHCH2OPO3H2
CHOHCHOH
CHOH
CHO
CH2OPO3H2
+O=
HO CHCCH2OH
HC OHCH2OPO3H2
TPPtransketolase
e.g.
xyulose-5-phosphate ribose-5-phosphate
sedoheptulose-7-phosphate
glyceraldehyde phosphate
10% of carbohydrate metabolized this way. This pathway provides pentoses for RNA and DNA synthesis and NADPH for the biosynthesis of fatty acids and other endogenous reactions.
Catalytic Mechanism – formation of adduct with C2 of thiazole ring
+H
+H
N S
CCH3
OH
NC
S
CHCH3
OH
NC
S
H
H+ + CH3 CHO
α-hydroxy-ethyl-TPP
H+
N S
CCH3 OH
C OO
3 2 H++NC
S
H
NC
S
CH3 C COOH
O
NC
Spyruvate
H+
CH3 C COOH
O
Deficiency • Signs of deprivation are mainly neurologic. • Thiamin needs are proportional to caloric intake because it is
essential for carbohydrate metabolism. • 0.5 mg/1000 calories plus 0.3 mg during pregnancy and
lactation. • Deficient in 20-30% of elderly and 40-50% of chronic
alcoholics. <2% healthy controls showed evidence of deficiency.
• Thiamin and alcoholism – Alcohol blocks conversion of thiamin à TPP – Alcohol decrease absorption, active transport, and storage – Alcohol increased fluid intake and urine flow à thiamin washout – Thiamin deficiency also involved in fetal alcohol syndrome
Deficiency Signs • Early signs: anorexia, nausea, vomiting, fatigue, weight loss,
nystagmus, tachycardia • Late signs: Beriberi
– Cardiac - increased heart size, edema (wet beriberi) – Cerebral - depression, irritability, memory loss, lethargy (dry beriberi) – GI tract - vomiting, nausea, weight loss
neurological - weakness, polyneuritis, convulsions. – Vary with age of patient, rapidity of onset, and severity of deficiency.
• Thiamin deprivation has been shown to cause oxidative stress, alter neurotransmitter metabolism, and cause dysfunction of the BBB, which may account for some of the neurological symptoms.
Beriberi (from Wikipedia) Edema
From aibolita.com
Wernicke-Korsakoff Syndrome • Wernicke encephalopathy (dilirium) and Korsakoff
dementia. • Neurological disorder resulting in impaired mental
functioning. • Seen in some alcoholics. • Also seen in an inborn error of metabolism in transketolase • Symptoms: confusion, memory loss, confabulation, psychotic
behavior. Overlap with symptoms of dry beriberi.
Thinning of Corpus callosum and enlargement of ventricles
Risk Factors
• Increased carbohydrate intake: total parenteral nutrition (IV for GI disorder), alcoholics
• Decreased absorption: ulcerative colitis, alcoholism
• Decreased intake: poor diet, geriatrics, breast fed infant from B1 deficient mother
• Alcoholism
Diagnosis of Deficiency State
• Increased levels of pyruvate and lactate in plasma
• Transketolase activity in RBC – most important technique
CHOHCHO
CH2OPO3H2
+O=
HOCHCCH2OH
HCOH
HCOH
HCOHCH2OPO3H2
CHOHCHOH
CHOH
CHO
CH2OPO3H2
+O=
HO CHCCH2OH
HC OHCH2OPO3H2
TPPtransketolase
e.g.
xyulose-5-phosphate ribose-5-phosphate
sedoheptulose-7-phosphate
glyceraldehyde phosphate
Use • Deficiency states – alcoholics • Acute alcoholism: give 100 mg IM or IV stat. This is a common
practice. • Thiamin responsive inborn errors of metabolism (next slide) • Alzheimer’s disease – little evidence for benefit (huge doses
used) (although 20% reduction of TPP levels was observed in patient brain – association, but may not be causative).
• Requirement: 0.5 mg/1000 cal. DV = 1.5 mg. Minimum intake 1 mg.
• Rarely needed as a single supplement; can be taken in a multivitamin.
• Nontoxic on oral administration; anaphylactic reactions have been observed in patients receiving repetitive parenteral doses.
• Stability: labile to alkaline pH and heat.
Thiamin Responsive Inborn Errors of Metabolism
Disease Defect
Wernicke-Korsakoff Transketolase
Maple syrup urine disease Failure to decarboxylate branched chain amino acids
Thiamin responsive megaloblastic anemia Thtr1 (transport)
Hyperalaninemia (elevated alanine in serum)
Pyruvate dehydrogenase
Hyperpyruvic acidemia Pyruvate dehydrogenase
OOH
ONADTPPCoASH
O
SCoA
Pyruvate Acetyl CoA
NH3O
O
Alanine
Transaminase(B6-dependent)
Pyruvate dehydrogenase
Dietary Sources • Cereal grains, lean meat (pork), eggs, yeast, nuts – Today, most white flour, rice and pastas are enriched to
bring thamin levels up (recall thiamin in polishings); other water soluble vitamins also enriched.
• However, thiaminase exists in some raw fish and shellfish and ferns, which can break down thiamin.
Image from vkool.com
OHS
N+
NH2N
N
Riboflavin (B2) - Structure OH
NNH
O
NOH
HOOH
N O
FMN = riboflavin monophosphateFAD = flavin adenine dinucleotide riboflavin coenzymes
riboflavin coenzymesFAD = flavin adenine dianucleotideFMN = riboflavin monophosphate
N
NNH
N
O
OCH3
CH3
CH2 CH CH CH CH2OHOH OH OH
gut mucosa liverriboflavin FMN FADATP ADP ATP ADP
Absorption
• Hydrolysis of coenzyme forms: riboflavin in most food is in protein complexes as coenzyme, so need hydrolysis, which occurs by the proteolytic activity of intestinal lumen.
• Active transport of free riboflavin: an ATP-dependent, carrier-mediated process in the proximal small intestine and colon.
Transport
• Transported as free riboflavin and FMN, both of which are bound to plasma protein in significant amounts.
• Specific binding proteins (RfBPs): found in plasma. • Cellular uptake: similar to enteric absorption,
carrier mediated. • Tissue distribution: mostly as FMN (60-95%) and
FAD (5-22%). High in liver, kidney and heart.
Function
• Serve as intermediates in transfers of e- in biological reduction-oxidation (redox) reactions, tissue respiration, H transfer as flavin containing enzyme.
• Coenzymes for > 100 enzymes. • Essential for metabolism of carbohydrates, amino
acids, and lipids
NNH
O
NR
N O
NH
NH
O
NR H
N O+2 H
-2 H
Oxidized - Yellow Reduced - colorless
Examples of Flavin-containing Enzymes • Succinate dehydrogenase (succinate à fumerate in TCA
cycle)
• Fatty acid acyl CoA dehydrogenase (β-oxidation of lipids)
• Cytochrome C reductase • NADPH-cytochrome C reductase • Cytochrome P450 reductase (drug metabolism) • Flavin-containing monooxygenase (drug metabolism) • Glutathione reductase – important in antioxidant activities
NADPH
NADP
FAD
FADH2
2GSH
GSSG
H2O2 or ROOH
2H2O or ROH + H2O
glutatione peroxidaseglutathionereductase
HO
OOH
OHO
OOH
O
succinate fumerate
SCoA
O
n SCoA
O
n
Health Related
• Protection against oxidative stress (antioxidant enzymes) • Reduce risk of vascular disease: inversely correlated
with homocysteine level, which is a risk factor • Anticarcinogenesis: cancer inversely correlated with
decreased antioxidant activity (more DNA oxidative damage)
• Deficiency protect against malaria: increase vulnerability of erythrocytes to lipid peroxidation, but malarial parasites are more susceptible than erythrocytes to oxidative stress.
Deficiency State
• Not usually seen in isolation but occurs in combination with other B vitamin deficiencies.
• Fatigue, cheilosis, glossitis, vascularization of cornea, dermatitis • Vegans and teenagers may be low in B2 if dairy intake is low • Low B2 intake may be a risk factor for cataract development • Alcoholics are at risk due to low intake and low absorption. • Other risk factors: phototherapy, exercise • Diagnosis: erythrocyte GSH reductase activity is a useful marker
for riboflavin status.
Use • For deficiency states. Is a component of most multivitamin mixtures. • May help in migraine headache prevention. Need 400 mg/day. • High intake associated with lower risk for cataracts and a 3mg
supplement reduced risk. • DV = 1.7 mg; no UL value (excess vitamin is efficiently eliminated
renally); nontoxic. • Average US diet contains 2 mg for males and 1.5 mg for females. • Will turn urine bright yellow in doses higher than DV. • Supplemented as multivitamin. • Labile to heat, light, and base.
Sources
• Milk, leafy veggies, meats, eggs, yeast
Image from medlineplus.gov
B6 - Structures
pyridoxamine (PN)
N
CH2OHHO
CH3
CH2NH2
N
CH2OHCH2OHHO
CH3
pyridoxine (P)
N
CH2OHHO
CH3
CHO
pyridoxal (PL)
Pyridoxine is a commonly used term for this vitamin, but all 3 are equally active, so vitamin B6 is a better term to use. Three phosphorylated forms are also present:
P PO4 PLP
FMNPNP
FMN
Coenzyme = pyridoxal-5-phosphate “PLP”
Transport
• Form Schiff base with lysine residue of proteins: albumin, hemoglobin
H3N
NH3
O
O
N
HO CH2OPCHO
+
N
HO CH2OPCHNR
Function
• Participates in over 140 enzymatic reactions by forming a Schiff base with the terminal amino group of lysine in the enzyme.
• Corresponds to ~4% of all enzymatic reactions known.
N
CH2OP
CH3
HOCH
O
R NH2
holoenzymeNCH3
HO CH2OPCHNR
PLP
PLP dependent enzyme
+
Function - Transamination (amino acids metabolism)
R C COOHO
α-keto acid #1
N
HO CH2OP
CH3
CHNenzyme
N
HO CH2OP
CH3
CHNCHR COOH
N
HO CH2OP
CH3
CHNCR COOHH
N
HO CH2OP
CH3
CHNH2
R CH COOHNH2
amino acid #1
+
+
H2O
2
2
+ R´ C COOHNH2
amino acid #2
+ R´ C COOHO
α-keto acid #2N
CH2
NH2
HO
CH3
CH2OP
N
HO
CH3
CH2OPCHO
PLPPNP
PLP
oxaloacetic acid alanineCH3 CH COOH
NH2
transaminasepyruvic acid
HOOC CH2 C COOHO
+
aspartic acid HOOC CH2 CH COOH
NH2+ CH3 C COOH
O
PLP
oxaloacetic acid alanineCH3 CH COOH
NH2
transaminasepyruvic acid
HOOC CH2 C COOHO
+
aspartic acid HOOC CH2 CH COOH
NH2+ CH3 C COOH
O
e.g. glutamate-aspartate transaminase
Function - Decarboxylation (synthesis of neurotransmitters and histamine from amino acids)
N
HO CH2OP
CH3
CHO
N
HO CH2OP
CH3
CHNCHR COOH
N
HO CH2OP
CH3
CHO
CH2RNH2+
decarboxylase
H2O
amino acid
R CH COOHNH2 + + CO2
HOOC CH2 CH2 C COOHH
NH2PLP
CO2
HOOC CH2 CH2 C HH
NH2
glutamic acid γ-amino butyric acid (GABA)
e.g.
N
HOC COOHH
NH2
5-hydroxytryptophan
N
HOCH2
NH2
5-hydroxytryptamine (serotonin)
CO2
PLP
PLP
CO2NN
CH2HCH
NH2
histamine
NNCH2
HC COOHNH2
histidine
B6 and Anti-Parkinson’s Drug (Levo-DOPA)
• B6 contraindicated in Levo-DOPA therapy because B6 enhances peripheral decarboxylation of Levo-DOPA to dopamine, which will not cross Blood Brain Barrier
• Larobec® (Roche) contains no pyridoxine and can be used if multivitamin supplementation is desired for patient on L-DOPA.
• The anti-Parkinsons’s drug Sinemet® contains levo-DOPA and carbidopa (a DOPA decarboxylase inhibitor) -- therefore, no interaction.
PLP
CO2 CH2 CH2
OH
NH2
HOHO
CH2HC COOHNH2
OH
DOPA DOPAMINE
Levo-DOPA
Chirality
Carbidopa
B6 and sulfur amino acid metabolism
C COOHCH2
H
NH2
CH2SCH3
OCH2
S CH2NH2
HCH2 COOCH
adenine
OHOH
-
OCH2
CH3 S CH2 CH2 COOCH
adenine
OHOH
H
NH2
-
C COOHCH2
H
NH2
H3C
C COOHCH2
H
NH2
HS
α-ketobutyrate
cysteine
cystathionine
PLP serine
methionine
S-adenosylmethionine
methyl acceptor
S-adenosylhomocysteine
THFA
N5-methylTHFA
methyl B12
hydroxy B12
ATP
C COOHCH2
H
NH2
CH2HS
homocysteine
C COOHCH2
H
NH2
CH2S
CH2 C COOHH
NH2
+
PLPcystathionineγ-lyase
Elevated homocysteine is an independent risk factor for cardiovascular disease and birth defects
B6 involvement in methionine formation (and S-adenosyl methionine) makes it indirectly involved in methylation (other involved vitamins folate B9 and B12): hence involvement in lipid metabolism and nucleic acid formation.
H2C C COOH
OH3C
Cystathionine synthase
MethylB12
B12N5-methylTHFA
THFA
Function - Tryptophan Metabolism to Serotonin and Niacin
NiacinN
COOH
COOH
NH2NH
tryptophanoxygenase
serotonintryptophan
COOH
NH2NH
HO
N-formylkynurenine
NH2NCHO
COOH
O
3-hydroxykynurenine
NH2
COOH
OHNH2
O
kynureninasePLP
COOH
NH2OH
3-hydroxy anthranilic acid
NNH2
HO
Htryp
decarboxylase
PLP
NiacinN
COOH
COOH
NH2NH
tryptophanoxygenase
serotonintryptophan
COOH
NH2NH
HO
N-formylkynurenine
NH2NCHO
COOH
O
3-hydroxykynurenine
NH2
COOH
OHNH2
O
kynureninasePLP
COOH
NH2OH
3-hydroxy anthranilic acid
NNH2
HO
Htryp
decarboxylase
PLP
Vitamin B3
Mechanisms of Pyridoxal/Pyridoxamine Reactions
CH2
N
C
N
HC
HC
N
N
B:
external imine internal imine
HO-
H2C
C
N
HN
OH
external carbinolamine
HC
HC
N
HN
HO-
OH
internal carbinolamine
H2C
N
NH2
OHC
N
O
NH2
• Hydrolysis of imine leads to incorporation of ‘oxygen atom’
• PLP enzymes control which carbon atom gets the oxygen atom by switching between the external imine and the internal imine. This accounts for their versatility.
Deficiency
• Symptoms: rash, peripheral neuritis, anemia and possible seizures.
• Not usually seen but could be induced by some treatment (iatrogenic)
• Isoniazid – also called isonicotinylhydrazide (INH), an antituberculosis drug, forms Schiff base with B6, leading to deficiency. Symptoms: neuritis and convulsion. Use 25-300 mg/d B6 for prevention.
N
CO
NH NH2
N
HCO
CH2OHHO
CH3
+
N
CO
NH N CHO CH2OH
H
CH3
NIsoniazid
Deficiency diagnosis: measure erythrocyte transaminase activities and plasma PLP levels
NN
HNNH2
Hydralazine (vasodilator)
Use • In isoniazid (INH) therapy • PMS (50-500 mg/d) - evidence is uneven. PLP is known to bind to
steroid receptors. • Certain inborn errors of metabolism (next slide) • Carpal tunnel syndrome - evidence is uneven. It seems to work for
some. A trial of B6 100-200 mg/d for 6 mos. may be worthwhile. In some trials vitamin B6 combined with lipoic acid worked – effects are modest! (numbness, tingling, weakness, and other problems in the hand)
Use – Cont’d • Chinese restaurant syndrome: may be related to large amount of intake
of monosodium glutamate (MSG), buy has not been shown in clinical trials. Studies suggest B6 deficiency renders the patients unable to metabolize MSG (to GABA neurotransmitter) – Symptoms: chest pain, flushing, headache, numbness or burning in or around the
mouth, sense of facial pressure or swelling, sweating
• Lower homocysteine levels (combined with B9 and B12) – cardiovascular disease risk factor
• Nausea and vomiting in pregnancy. – PremesisRx contains 75mg sustained release B6 (plus 12ug B12, 1mg folic
acid and 200mg calcium) or 25mg of generic B6 TID is less expensive. PremesisRx is contraindicated for patients on Fluorouracil (thymidylate synthase inhibitor, cancer drug) – FU side effects may be increased by PremesisRx. Not used in the USA any longer.
MSG GABA
B6-Responsive Inborn Errors of Metabolism
Name Symptoms Dose of B6 Problem
B6-dependent infantile convulsions
Clonic and tonic seizures
10-25 mg/day
Defective glutamic acid decarboxylase; possible GABA depletion
B6-responsive anemia
Microcytic, hypochromic anemia
100 mg/day
Defective hemoglobin synthesis
Xanthurenic acidurea Mental retardation 25-100 mg/day Defective tryptophan metabolism due to faulty kyureninase, xanthurenic acid spills into urine
Homocystinurea Mental retardation Early heart disease
25-500 mg/day Defective cystathionine synthetase à homocysteine appears in urine
Cystathionurea Mental retardation 25-500 mg/day Defective cystathionase
Homocysteine CystathionineCystathionine
Synthetase CystathionaseCysteine + ⍺-ketobutyrate
Requirement, Toxicity, Sources • DV = 2 mg; UL = 100 mg. • > 200 mg/day can decrease prolactin levels; > 1-2 g/day
can cause serious neuropathy by an unknown mechanism. • Recommendation: avoid long term use in doses above 200
mg. • Sources: milk, meats, legumes, tuna, whole grains, beans • Pyridoxine is stable; pyridoxal could be lost during cooking
(Schiff base formation and oxidation)
Image from vkool.com
