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 (%)