historical context of vitamin b 12 pernicious anemia –megaloblastic anemia –neuropathy:...
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Historical Context of Vitamin B12Historical Context of Vitamin B12
• Pernicious anemia– Megaloblastic anemia– Neuropathy: particularly degeneration
of spinal cord– Universally fatal– Extrinsic factor from liver
• Patients were not producing enough– Gastric acid to denature R protein– Intrinsic factor
• Pernicious anemia– Megaloblastic anemia– Neuropathy: particularly degeneration
of spinal cord– Universally fatal– Extrinsic factor from liver
• Patients were not producing enough– Gastric acid to denature R protein– Intrinsic factor
Structure of Vitamin B12Structure of Vitamin B12
• Cobalamins– Corrin ring contains central cobalt
atom
• Adenosylcobalamin
• Methylcobalamin
• Cobalamins– Corrin ring contains central cobalt
atom
• Adenosylcobalamin
• Methylcobalamin
Absorption & Transport of Vitamin B12Absorption & Transport of Vitamin B12
• Receptors on ileal mucosal cells
• Transcobalamin II
• Portal circulation
• Receptor on the cell surface
• Endocytosis and lysosomal degradation of the complex
• Reduction of cobalt
• Cytosolic methylation
• Mitochondrial adenosylation
• Receptors on ileal mucosal cells
• Transcobalamin II
• Portal circulation
• Receptor on the cell surface
• Endocytosis and lysosomal degradation of the complex
• Reduction of cobalt
• Cytosolic methylation
• Mitochondrial adenosylation
Role of Ascorbate in the Degradation of TyrosineRole of Ascorbate in the Degradation of Tyrosine
• Oxidation of p-hydroxyphenylpyruvate– Maintain Cu2+?
• Homogentisate oxidation– Maintain Fe2+?
• Synthesis of epinephrine
• Bile acid synthesis (7-hydroxylase)
• Enhancement of iron absorption
• Antioxidant
• Oxidation of p-hydroxyphenylpyruvate– Maintain Cu2+?
• Homogentisate oxidation– Maintain Fe2+?
• Synthesis of epinephrine
• Bile acid synthesis (7-hydroxylase)
• Enhancement of iron absorption
• Antioxidant
Incubation 2: Transfer to a Incubation 2: Transfer to a microtiter well coated with microtiter well coated with biotin-BSA ( )biotin-BSA ( )
Incubation 1:Incubation 1:Biotin incubatedBiotin incubatedwith HRP-avidinwith HRP-avidin
Detection of Biotin ( ) by HRP-Avidin ( )Detection of Biotin ( ) by HRP-Avidin ( )BB
BB
BB BB BB BB BBBBBBBBBBBB BBBB
BB
BBBB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
Transfer
BBBB
Standard Curves for BiotinStandard Curves for Biotin
0.00.0
0.20.2
0.40.4
0.60.6
0.80.8
1.01.0
1.21.2
1.41.4
00 300300 600600 900900 1,2001,200 1,5001,500 1,8001,800
Ab
sorb
an
ce
Ab
sorb
an
ce
Avidin-binding Substance (pmol/L)Avidin-binding Substance (pmol/L)
Biotin CatabolitesBiotin Catabolites
BisnorbiotinBisnorbiotin
OO
CC
SS
HCHC
H2CH2C
HNHN
CHCH
CHCH
NHNHBiotin SulfoxideBiotin Sulfoxide
OO
- (CH2)4-C- (CH2)4-C
OO
OHOH
H2CH2C
OO
CC
SS
HCHC
HNHN
CHCH
CHCH
NHNH
- (CH2 )2 - C- (CH2 )2 - C
OO
CH3CH3
Bisnorbiotin methyl ketoneBisnorbiotin methyl ketone
H2CH2C
OO
CC
SS
HCHC
HNHN
CHCH
CHCH
NHNH
- (CH2)4-C- (CH2)4-C
OO
OHOH
Biotin sulfoneBiotin sulfone
OO OO
H2CH2C
OO
CC
SS
HCHC
HNHN
CHCH
CHCH
NHNH
- (CH2)2 - C- (CH2)2 - C
OO
OHOH
00
10001000
20002000
30003000
40004000
50005000
00 55 1010 1515 2020 2525 3030 3535
Ra
dio
ac
tiv
ity
(d
pm
)R
ad
ioa
cti
vit
y (
dp
m)
Retention Time (minute)Retention Time (minute)
BiotinBiotin
BisnorbiotinBisnorbiotin
Biotin SulfoxideBiotin Sulfoxide
HPLC of Radiolabeled Biotin AnalogsHPLC of Radiolabeled Biotin Analogs
Urinary Analogs Analyzed Against Authentic Standards
Urinary Analogs Analyzed Against Authentic Standards
353530302525202015151010550000
55
1010
1515
2020
2525
Retention Time (min)Retention Time (min)
Avi
din
-bin
din
g S
ub
stan
ces
Avi
din
-bin
din
g S
ub
stan
ces
(
pm
ol/
mg
cre
atin
ine)
(
pm
ol/
mg
cre
atin
ine)
un
kno
wn
#1
un
kno
wn
#2
bio
cy
tin
bio
cy
tin
un
kno
wn
#5
biotinbiotin
}
}
}
}
}
? d & l? d & l
biotin biotinsulfoxidesulfoxide
}
bisnorbiotinbisnorbiotin
}
}
met
hy
l ke
ton
em
eth
yl k
eto
ne
Mole Percentage of Urinary Biotin and Mole Percentage of Urinary Biotin and Metabolite in 10 SubjectsMetabolite in 10 Subjects
Mole Percentage of Urinary Biotin and Mole Percentage of Urinary Biotin and Metabolite in 10 SubjectsMetabolite in 10 Subjects
Unk1Unk1Unk1Unk1 BSOBSOBSOBSO BNBBNBBNBBNB Unk2Unk2Unk2Unk2 Unk3Unk3Unk3Unk3 Unk4Unk4Unk4Unk4 BiotinBiotinBiotinBiotin Unk5Unk5Unk5Unk50000
20202020
40404040
60606060
80808080
Biotin MetabolitesBiotin MetabolitesBiotin MetabolitesBiotin Metabolites
Bio
tin
or
Met
abo
lite
B
ioti
n o
r M
etab
oli
te
(mo
l/10
0 m
ol)
(mo
l/10
0 m
ol)
Bio
tin
or
Met
abo
lite
B
ioti
n o
r M
etab
oli
te
(mo
l/10
0 m
ol)
(mo
l/10
0 m
ol)
Mole Percentage of Serum Biotin Mole Percentage of Serum Biotin and Metabolites in 15 Subjectsand Metabolites in 15 Subjects
Mole Percentage of Serum Biotin Mole Percentage of Serum Biotin and Metabolites in 15 Subjectsand Metabolites in 15 Subjects
Bio
tin
or
Met
abo
lite
(m
ol/
100
mo
l)B
ioti
n o
r M
etab
oli
te
(mo
l/10
0 m
ol)
BSOBSO BNBBNB BiotinBiotin All UnknownsAll Unknowns00
2020
4040
6060
8080
100100
Biotin MetaboliteBiotin Metabolite
GCRC Study DesignGCRC Study Design
egg white dietegg white diet (avidin)(avidin)
non-biotin vitamin supplementnon-biotin vitamin supplementbiotin supplementbiotin supplement
Blood and urine collectionBlood and urine collection
-10 0 3 7 10 14 17 20 30-10 0 3 7 10 14 17 20 30
Urinary Excretion of BiotinUrinary Excretion of Biotin
00
10102020
30304040
5050
60607070
8080N
. R.
N. R
.
Study DayStudy Day
* p< 0.008 by range test after ANOVA
Bio
tin
Bio
tin
(nm
ol/2
4 h
)(n
mo
l/24
h)
** ** ** ** ** **
00 33 77 1010 1414 1717 2020
Mean ± SD
Serum Concentration of BiotinSerum Concentration of Biotin
00
200200
400400
600600
800800
Ser
um
Bio
tin
S
eru
m B
ioti
n Mean ± SD
(pm
ol/
L)
(pm
ol/
L)
00 33 77 1010 1414 1717 2020
N. R
.N
. R.
Study DayStudy DayStudy DayStudy Day
Biotin-dependent CarboxylasesBiotin-dependent Carboxylases
Acetyl-CoAAcetyl-CoA Malonyl-CoAMalonyl-CoA
Cytosol and Mitochondria:Cytosol and Mitochondria:
PyruvatePyruvate Oxaloacetic acidOxaloacetic acid
Propionyl-CoAPropionyl-CoA Methylmalonyl-CoAMethylmalonyl-CoA
3-Methylcrotonyl-CoA3-Methylcrotonyl-CoA 3-Methylglutaconyl-CoA3-Methylglutaconyl-CoAMCCMCC
PCCPCC
PCPC
ACCACC
Mitochondria:Mitochondria:
Biosynthesis of HolocarboxylasesBiosynthesis of Holocarboxylases
Apocarboxylase + biotin + ATPApocarboxylase + biotin + ATP
Holocarboxylase + AMP + pyrophosphateHolocarboxylase + AMP + pyrophosphate
Holocarboxylase synthetase
Holocarboxylase synthetase
Mechanism of Biotin-dependent CarboxylaseMechanism of Biotin-dependent Carboxylase(Pyruvate carboxylase)(Pyruvate carboxylase)
Biotinidase is a Digestion & Salvage Enzyme
Biotinidase is a Digestion & Salvage Enzyme
E-BiotinE-BiotinBiotinidaseBiotinidase
ProteasesProteases
Peptides+
Biotin
Peptides+
Biotin
3-methylcrotonyl-CoA3-methylcrotonyl-CoA
3-Methylcrotonyl-CoA 3-Methylcrotonyl-CoA CarboxylaseCarboxylase
3-Methylcrotonyl-CoA 3-Methylcrotonyl-CoA CarboxylaseCarboxylase
3-methylglutaconyl-CoA3-methylglutaconyl-CoA
3-HIA3-HIA
leucineleucine
Urinary Excretion of 3-HIAUrinary Excretion of 3-HIA
00
100100
200200
300300
400400
500500
600600
3-H
ydro
xyis
ova
leri
c A
cid
3-H
ydro
xyis
ova
leri
c A
cid
(µm
ol/
24 h
)(µ
mo
l/24
h)
N. R
.N
. R.
**
** **** **
00 33 77 1010 1414 1717 2020
* p< 0.008 by range testafter ANOVA
**
Mean ± SD
Study DayStudy DayStudy DayStudy Day
MalformationMalformation VitaminVitamin Trace-ElementTrace-Element GroupGroup Group Group
Neural-tube defectNeural-tube defect 0 0 6 6
Congenital hydrocephalus 0Congenital hydrocephalus 0 2 2
Cleft palateCleft palate 0 0 2 2
Limb-reduction defect Limb-reduction defect 1 1 5 5
Cleft lipCleft lip 4 4 3 3(with or without cleft palate)(with or without cleft palate)
Czeizel & Dudas “Prevention of the first occurrence of neural-tube defects byCzeizel & Dudas “Prevention of the first occurrence of neural-tube defects by
periconceptional vitamin supplementation”periconceptional vitamin supplementation” NEJM, 326: 1832 - 5, 1992.NEJM, 326: 1832 - 5, 1992.
MalformationMalformation VitaminVitamin Trace-ElementTrace-Element GroupGroup Group Group
Neural-tube defectNeural-tube defect 0 0 6 6
Congenital hydrocephalus 0Congenital hydrocephalus 0 2 2
Cleft palateCleft palate 0 0 2 2
Limb-reduction defect Limb-reduction defect 1 1 5 5
Cleft lipCleft lip 4 4 3 3(with or without cleft palate)(with or without cleft palate)
Czeizel & Dudas “Prevention of the first occurrence of neural-tube defects byCzeizel & Dudas “Prevention of the first occurrence of neural-tube defects by
periconceptional vitamin supplementation”periconceptional vitamin supplementation” NEJM, 326: 1832 - 5, 1992.NEJM, 326: 1832 - 5, 1992.
Urinary Excretion of BiotinUrinary Excretion of Biotin
n.s.n.s.
p < 0.05p < 0.05
p < 0.003p < 0.003
controlcontrol earlyearly latelate00
2020404060608080100100120120140140160160
Bio
tin
Exc
reti
on
(p
mo
l/mg
cre
at)
Bio
tin
Exc
reti
on
(p
mo
l/mg
cre
at)
Urinary Excretion of 3-HIAUrinary Excretion of 3-HIA3H
IA E
xcre
tio
n(µ
mo
l/24
h)
3HIA
Exc
reti
on
(µm
ol/
24 h
)
00
5050
100100
150150
200200
250250
300300
controlcontrol earlyearly latelate
p < 0.0001
p < 0.0001
Biotin Treatment Decreases 3-HIA Excretion
Biotin Treatment Decreases 3-HIA Excretion
Ch
ang
e in
Uri
nar
y 3-
HIA
C
han
ge
in U
rin
ary
3-H
IA
(mm
ol/
mo
l cr
eati
nin
e)(m
mo
l/m
ol
crea
tin
ine)
-15-15-15-15
-10-10-10-10
-5-5-5-5
0000
5555
PlaceboPlacebo BiotinBiotin
p = 0.001
Late BiotinLate BiotinLate BiotinLate Biotin
Early BiotinEarly BiotinEarly BiotinEarly Biotin
Late PlaceboLate Placebo
Early PlaceboEarly Placebo
Marginal Biotin Deficiency is Very Teratogenic in ICR Mice Marginal Biotin Deficiency is Very Teratogenic in ICR Mice
• No signs or symptoms in the dam
• Normal dam & fetal weight gain
• Resorptions < 6% and global disruption was not seen
• High rates of skeletal malformations
• No signs or symptoms in the dam
• Normal dam & fetal weight gain
• Resorptions < 6% and global disruption was not seen
• High rates of skeletal malformations
Whole Fetal SkeletonWhole Fetal Skeleton
Increase in Limb Malformations with Increase in Limb Malformations with Increasing Egg White Content Increasing Egg White Content
Increase in Limb Malformations with Increase in Limb Malformations with Increasing Egg White Content Increasing Egg White Content
00
2020
4040
6060
8080
100100 Forelimb HypoplasiaHindlimb Hypoplasia
Control 0 1 1.3 2 3 5 10 25Control 0 1 1.3 2 3 5 10 25
Mal
form
atio
ns
(%)
Mal
form
atio
ns
(%)
Dietary Egg White (%)Dietary Egg White (%)