Recent Advances in Vitamin K Metabolism

3rd International Conference on Nutrition and Food Science(Nutritional Science-2014)Track 1: Nutrition and Basic Science14:10-14:30, September 23, 2014Committee Room 1-2Palacio de Congresos de Valencia, Spain

Kobe Pharmaceutical UniversityToshio Okano

Dept. Hygienic SciencesKobe Pharmaceutical Univ.

O

O

O

O

Naturally occurring vitamin K

Plant origin（Major dietary

source）

PK (phylloquinone: vitamin K1）

Bacterial origin（Minor dietary

MK-4 (menaquinone-4: vitamin K2)MK-7 O

O

Plasma vitamin K concentrations of healthy Japanese women

phylloquinone (ng/mL) MK-4 (ng/mL)1.74±1.29 (0.13〜8.83) 0.10±0.19 (n.d.〜1.44)

（Minor dietary

source） MK-7 (menaquinone-7)

Tsugawa N, Okano T, et al, Am J Clin Nutr, 2006;83:380-386.

Tissue concentrations of animals and humansMK-4＞＞ phylloquinone

Amounts of dietary intake and plasma concentrations phylloquinone＞＞ MK-4

Is phylloquinone converted into MK-4 in the body ?

The aim of our study is to examine the above issues in animals and humans.

What is the physiological significance of this conversion and what functions dose MK-4 have, and can we develop MK-4

analogues for clinical use ? Suhara Y, Okano T, et al, J Med Chem, 2011;54:4269-4273

Suhara Y, Okano T, et al, J Med Chem, 2011;54:4918-4922

Suhara Y, Okano T, et al, J Med Chem, 2012;55;1553-1558

Tissue concentrations of PK and MK-4 in mice fed a conventional laboratory chow diet

0

5

10

15

20

25

30

35

40

PK C

onc.（pm

ol/g）

PKPKPKPK

PKPKPKPK epoxideepoxideepoxideepoxide

0

200

400

600

800

1000

1200

1400

1600

1800

2000

MK-4 C

onc.（pm

ol/g）

MK-4

MK-4 epoxide

×100

Okano T. et al., JBC 2008;283:11270-11279

LC-MS and NMR

PK

Authentic vitamin KsMK-4

MK-4 fraction

Inte

ns

ity,

cp

s

MK-4

Identification of MK-4 from brain of mice by LC-MS/MS

MK-4

MK-4 epoxide

PK epoxide

Time,min

PKPK

epoxide

Time,min

PKMK-4

epoxidePK

epoxide

Inte

ns

ity,

cp

s

MK-4 epoxide PK

PK epoxide

H-5,88.06 H-6,7

7.67

H-2’,6’,10’,14’5.03

H-1’3.45

CH3（（（（C-2））））2.17

Authentic MK-4

Identification of MK-4 from brain of mice by1H NMR spectrometryO

O

1 2

345

6

7

8

2’ 6’ 10’ 14’

1’

7.67 5.03

012345678δδδδ（（（（ppm））））

H-5,88.06

H-6,77.68

H-2’,6’,10’,14’5.08

H-1’3.35

MK-4 fraction CH3（（（（C-2））））2.17

1 w 24 hr

PK-d7

12 hr

C57BL/6J mice7-weeks old

Fast Sacrifice

0

Oral administration

Cerebra

Experimental design

Experiment

7-weeks oldn=200

Analyses by LC-MS/MS and 2H NMR

Isolation and purification of MK-4-d7

O

O 3

PK-d7

D

D

D

D

CD3

O

O 3

MK-4 -d7

D

D

D

D

CD3？？？？

cps）

4.5e4

3.0e4Inten

sity（

cps）

0

Authentic MK-4-d7

MK-4-d7 fraction

194.3452.5

Identification of MK-4-d7 from brain of mice byLC-MS/MS

3.5e4

1.5e4

（（（（m/z））））

Inten

sity（

cps

100 200 300 400 50000

O

O

CH3

CD3

D

D

D

D

m/z：194

194.4452.6

+ H

2H-5,8 C2H

Authentic MK-4-d7

O

O

CD3

D

D

D

D

1 2

345

67

8MK-4-d7

Identification of MK-4-d7 from brain of mice by2H NMR spectrometry

H-5,88.10

2H-6,77.70

C2H3

2.14

012345678δδδδ（（（（ppm））））

2H-6,77.68

2H-5,88.08

MK-4-d7fractionC2H3

2.14

Not only PK-d7 but also MK-4-d9 and MK-7-d7 are converted into MK-4-d7 and accumulate in brain of mice

O

O 3

D

D

D

D

CD3PK-d7

O

O 3

D

D

D

D

CD3

D D

MK-4-d9 MK-7-d7O

O 6

D

D

D

D

CD3

24 hr12 hr

Fast Sacrifice

0

C57BL/6J ♀mice

8 weeks old

O

O 3

D

D

D

D

CD3 MK-4-d7

Single peroral administration (10 µg/kg)0

20

40

60

80

100

120

vehicle PK-d7 MK-4-d9

pmol/

gof b

rain

MK-7-d7

MK-4MK-4 epoxide

MK-4-d7

MK-4-d7 epoxideEndogenous MK-4

Phylloqunone is converted into MK-4 via integral side-chain removal

Unsaturation of side chain

O

O 3

O

O 3

O

O 3

O

O 3

DDDD

MK-4-d9CD3

D

D

D

D

D D

CD3

D

D

D

D

D D

K1-d9

D DD D

CD3

D

D

D

D

CD3

D

D

D

D

OO Exchange of OO

D DD D

MK-4-d7CD3

D

D

D

D

CD3

D

D

D

D

CD3

D

D

D

D

CD3

D

D

D

D

O

O

O

OPhytyl side chain

Geranylgeranylside chain

33

3

PP

PO O

O-

OP :

PO O-

O-

OP

PO O

O-

OP :

PO O-

O-

OPK3

Exchange of

side chain

O

O 3

O

O 3

K vitamins are converted into MK-4 and accumulate in

tissuesWhere does this conversion take

place?Following four routes for the Following four routes for the conversion of PK or K3 into MK-4,

１．Oral route２．Enteral route３．Intravenous route４．Intra-cerebroventricular

routewere examined in mice.

1 w 24 hr

K1-d7, K3-d8

12 hrC57BL/6J mice

7 weeks old

Fast Sacrifice

0

Oral(1), enteral(2), intravenous(3) or intra-cerebroventricular(4) dose

Cerebra

LC-APCI//MS

Experimental design

A single dose of 10 μmol/kg BW for (1), (2), (3) At 0.1 μmol/Kg BW for (4)

O

O 3

K1-d7

D

D

D

D

CD3

O

O 3

MK-4-d7

D

D

D

D

CD3

O

O K3-d8

D

D

D

D

CD3

D

Concentrations of MK-4 in cerebra of mice

0

50

100

150

200

Co

nc

en

tra

tio

n

（（ （（p

mo

l/g）） ））

Vehicle K3-d8PK-d Vehicle K3-d8PK-d7

0

100

200

300

MK-4 MK-4 epo MK-4-d7 MK-4-d7 epo

Endogenous MK-4 Converted MK-4

oral entero

Vehicle K3-d8PK-d7Vehicle K3-d8PK-d7

Vehicle K3-d8PK-d7

0

50

100

150

200

250

Co

nc

en

tra

tio

n

（（ （（p

mo

l/g）） ））

Vehicle K3-d8PK-d70

50

100

150

200

250intravenous Intra-cerebroventricular

Okano T, et al, J Biol Chem, 2008; 283:11270-11279

Four Sites Cannulation experiments

Bile duct Portal vein

Inferior vena cava

Thoracic Lymph duct

heartheartheartheart

liver

serum (IVC)

Lymph(TLD)

Po K1-d7

0 1 62 3 4 5 (h)

sacrificeWistar rat

Bile duct Portal vein

intestineintestineintestineintestine

Bile (BD) Serum (PV)

6080100181.10525

[M+H]+

Authentic MD-d8 173.10173.08173.06173.04173.02173.00020406080100

173.05982[M+H]+

Authentic MD

(m/z )Relative Abundance (%

)ODD CD3

OORelative Abundance (%

)0102030405060

0 1 2 3 4 5 6Concentration(pmol/g) bileSerum from IVCSerum from PVLymph

0.030.040.05 MK-4-d7

K1-d7

oncentration(pmol/g)

high resolution mass spectrometry

(HR-MS)

bileSerum from IVCSerum from PVLymph

Concentrations of K1-d7, MK-4-d7 and MD-d7

180.06 180.08 180.10 180.12 180.14 180.16020406080100

180.10172[M+H]+

181.16181.14181.12181.10181.08181.060204060[M+H]+

MD fraction from lymphOODDD D CD3OD D 3D

(m/z )(m/z )

Relative Abundance (%)

Relative Abundance (%)00.010.02

0 1 2 3 4 5 6Time after administration (h)00.40.81.2

0 1 2 3 4 5 6Time after administration (h)Concentration (pmol/mL) MD-d7Concentration

bileSerum from IVCSerum from PVLymph MD-d7

400

con

cen

tra

tio

n(p

mo

l/m

L)

600

4 c

on

cen

tra

tio

n(p

mo

l/m

L)

6

8

10

12

18

24

MD

co

nce

ntr

ati

on

(pm

ol/

mL)

K1 MK-4 MD

Time course changes in serum concentrations of K1, MK-4 and MD in humans orally given K1 capsules (40 mg)

(n=5) (n=5) (n=5)

200

0 1 2 3 4 5 6

K1

con

cen

tra

tio

n

0 1 2 3 4 5 6

MK

-4 c

on

cen

tra

tio

n(p

mo

l/m

L

0

2

4

0

6

12

0 1 2 3 4 5 6

MD

co

nce

ntr

ati

on

(pm

ol/

mL

Time after administration (h)Time after administration (h)Time after administration (h)

0

GGPP

MK-4-d2

Route 1

Conversion pathway of MD-d3 to MK-4-d3 demonstrated by HR-MS and

1H NMR analyses (Route 2)

MD-d

GGPPMK-4-d3

Route 2

MD-d3

Hirota Y, Okano T, et al, J Biol Chem, 2013; 288:33071-33080

K1

Removal of side chainSide-chain cleavage

enzyme

MD (K3)(quinone form))

Lymph and blood stream

(intestine)

MD(K3) is a catabolic product of oral phylloquinone (K1) in the intestine and

a circulating precursor of tissue MK-4 (K2) in mammals

Prenylation enzyme

Geranylgeranyl pyrophosphate

MK-4 (K2)

Reductase (tissues)

℗-℗℗-℗：

Hirota Y, Okano T, et al, J Biol Chem, 2013; 288:33071-33080

MD (K3)(hydroquinone form))

New paradigm of the metabolic activation of

vitamin K in brain and bone

Biological actions

O

O 3

O

phylloquinone(PK)

GGCX, SXR/PXR, PKA/PKC

brain

Menaquinone-4（（（（MK-4））））

O

O 3

active metabolite

O

O

Foods

OR n-1

R:

menaquinones(MKn)

Menadione(MD)

bone

O

O 3

GGCX, SXR/PXR, PKA/PKC

Biological actions

O ？

What is the enzyme(s) involved in MK-4

biosynthesis in mammals ?

phylloquinone(PK)

O

OMenaquinone-4（（（（MK-4））））

O

O 3

foods

OR n-1

R:

Enzyme(s)

active metabolite

Menadione(MD)

menaquinones(MKn)

Biosynthesis of menaquinones in Escherichia. coli

menB

menF

menEmenCmenD

COO-

OHO COO-

Chorismate

O-Succinylbenzoyl-CoA 1,4-Dihydroxy-2-napthol-CoA

O

-OOCOH

COO-

Isochorismate

OH

OH

S-CoA

O

COO-

S-CoA

O

O

n

P2O73- P2O7

3-

nH

OH

OH

menG

yfbB

1,4-Dihydroxy-2-napthanoate Demethlmenaquinone

hydroquinone（（（（DMK-n））））

Menaquinone-nhydroquinone（（（（MK-n））））

Menaquinone-n

（（（（MK-n））））

O-

OH

OH OCO2

nP2O7

n

OH

OH

H

O

O n

menA

Alignment of the amino acid sequence of Ubia(E.coli), COQ2(Homo-sapiens), Men A(E.coli)

and UBIAD1(Homo-sapiens) ubiA_[Escherichia_coli] 1 ------------------------------------------------------------

COQ2_[Homo_sapiens] 1 MLGSRAAGFARGLRAVALAWLPGWRGRSFALARAAGAPHGGDLQPPACPEPRGRQLSLSA

_menA_[Escherichia_coli] 1 ------------------------------------------------------MTEQQI

UBIAD1_[Homo_sapiens] 1 ----------------------------MAASQVLGEKINILSGETVKAGDRDPLGNDCP

ubiA_[Escherichia_coli] 1 -MEWSLTQNKLLAFHRLMRTDKPIGALLLLWPTLWALWVATPG--VPQLWILAVFVAGVW

COQ2_[Homo_sapiens] 61 AAVVDSAPRPLQPYLRLMRLDKPIGTWLLYLPCTWSIGLAAEPGCFPDWYMLSLFGTGAI

_menA_[Escherichia_coli] 7 SRTQAWLESLRPKTLPLAFAAIIVGTALAWWQGHFDPLVALLALITAGLLQILSNLANDY

UBIAD1_[Homo_sapiens] 33 EQDRLPQRSWRQKCASYVLALRPWSFSASLTPVALGSALAYRSHGVLDPRLLVGCAVAVL

ubiA_[Escherichia_coli] 58 LMRAAGCVVNDYADRKFDGHVKRTANRPLPSGAVTEKEARALFVVLVLISFLLVLTLNTM

COQ2_[Homo_sapiens] 121 LMRGAGCTINDMWDQDYDKKVTRTANRPIAAGDISTFQSFVFLGGQLTLALGVLLCLNYY

_menA_[Escherichia_coli] 67 GDAVKGSDKPDRIGPLRGMQKGVITQQEMKRALIITVVLICLSGLALVAVACHTLADFVG

UBIAD1_[Homo_sapiens] 93 AVHGAGNLVNTYYDFSKGIDHKKSDDRTLVDRILEPQDVVRFGVFLYTLGCVCAACLYYL

ubiA_[Escherichia_coli] 118 T------ILLSIAALALAWVYPFMKR---YTHLPQVVLGAAFGWSIPMAFAAVS-ESVPL ubiA_[Escherichia_coli] 118 T------ILLSIAALALAWVYPFMKR---YTHLPQVVLGAAFGWSIPMAFAAVS-ESVPL

COQ2_[Homo_sapiens] 181 S------IALGAGSLLLVITYPLMKR---ISYWPQLALGLTFNWGALLGWSAIKGSCDPS

_menA_[Escherichia_coli] 127 F------LILGGLSIIAAITYTVGNRPYGYIGLGDISVLVFFGWLSVMGSWYLQAHTLIP

UBIAD1_[Homo_sapiens] 153 SPLKLEHLALIYFGGLSGSFLYTGGIGFKYVALGDLIILITFGPLAVMFAYAIQVGSLAI

ubiA_[Escherichia_coli] 168 SCWLMFLANILWAVAYDTQYAMVDRDDDVKIGIKSTAILFGQY-DKLIIGILQIGVLALM

COQ2_[Homo_sapiens] 232 VCLPLYFSGVMWTLIYDTIYAHQDKRDDVLIGLKSTALRFGEN-TKPWLSGFSVAMLGAL

_menA_[Escherichia_coli] 181 ALILPATACGLLATAVLNINNLRDINSDRENGKNTLVVRLGEVNARRYHACLLMGSLVCL

UBIAD1_[Homo_sapiens] 213 FPLVYAIPLALSTEAILHSNNTRDMESDREAGIVTLAILIGPT--FSYILYNTLLFLPYL

ubiA_[Escherichia_coli] 227 AIIGELNGLGWGYYWSILVAGALFVYQQKLIANREREACFKAFMNNNYVGLVLFLGLAMS

COQ2_[Homo_sapiens] 291 SLVGVNSGQTAPYYAALGAVGAHLTHQIYTLDIHRPEDCWNKFISNRTLGLIVFLGIVLG

_menA_[Escherichia_coli] 241 ALFNLFSLHSLWGWLFLLAAPLLVKQARYVMREMDPVAMRPMLERTVKGALLTNLLFVLG

UBIAD1_[Homo_sapiens] 271 VFSILATHCTISLALPLLTIPMAFSLERQFRSQAFNKLPQRTAKLNLLLGLFYVFGIILA

ubiA_[Escherichia_coli] 287 -YWHF----------------

COQ2_[Homo_sapiens] 351 NLWKEKKTDKTKKGIENKIEN

_menA_[Escherichia_coli] 301 IFLSQWAA-------------

UBIAD1_[Homo_sapiens] 331 PAGSLPKI-------------

Search for the enzyme(s) responsible for the MK-4 synthesis

E.coliOH

OH

O-

O

1,4-Dihydroxy-2-naphthoate

（（（（DHNA））））

nH

OH

OH

Demethylmenaquinone-n

hydroquinone（（（（DMK-n））））

MenA

Menaquinone-n

（（（（MK-n））））

MenG

H

O

On

nH

Menaquinone-n biosynthesis in E.coli

MenA human homologs

Chromosome 1 11.25 MMenA Prenylation domain 311 a.a

O

O 3

MK-4O

O

+3

P P

GGPPMD

UBIAD1 orCOQ2 ?

338 a.a

384 a.a

Chromosome 1 11.25 M

Chromosome 4 84.40 M

MenA

UbiA prenyltransferasedomain containing 1

（（（（UBIAD1））））

CoenzymeQ2 homolog,prenyltransferase

（（（（COQ2））））

Prenylation domain

Prenylation domain

311 a.a

Prenylation domain

Co

nce

ntr

ati

on

(p

mo

l/m

g p

rote

in)

Conversion of K3-d8 or MK-4-d12 to MK-4-d7 in siControl-, siUBIAD1- or siCOQ2-transfected MG-63 cells

Co

nce

ntr

ati

on

(p

mo

l/m

g p

rote

in)

MD-d8 MD-d8MD-d8MD-d8

40

50

60

70

80

90

Co

nce

ntr

ati

on

(

pm

ol/

mg

pro

tein

) MK4-d7

MK4-d7 epoxide

∗∗∗

∗∗∗

Conversion of PK-d7 and MK-4-d12 into MK-4-d7 in Sf9 cells transfected

with siControl or pcDNA3.3-UBIAD1(UBIAD1 expression vector)

EtOH MD-d8 PK-d7 MK-4-d12

0

10

20

30

40

Co

nce

ntr

ati

on

(

pm

ol/

mg

pro

tein

)

EtOH MD-d8 PK-d7 MK-4-d12

Sf9-Control Sf9-UBIAD1 expression vector

∗∗∗

Values are means and s.e.m. (n=6). Three asterisks, P<0.001 versus control-infected Sf9 cells with the same

compound treatment.

UB

IAD

1 a

cti

vit

y

(pm

ol/m

g p

rote

in/m

in-1

)

20

25

30

35

40

30

40

50

Sf9-control

Sf9-UBIAD1

UB

IAD

1a

cti

vit

y(p

mo

l/m

g p

rote

in/m

in-1

)

Substrate: K3-d8 (1 µµµµM)

Sf9-control

Sf9-UBIAD1

MK-4 biosynthetic activity of UBIAD1 in microsomes prepared from

UBIAD1 baculovirus-infected Sf9 cells with geranylgeranyl

pyrophosphate (GGPP) and MD

UB

IAD

1

(pm

ol/m

g p

rote

in/m

in

0

5

10

15

20

Substrate: MD-d8

0.01 0.1 1 (µµµµM)0

10

20

0 0.01 0.1 1 (µµµµM)

GGPP

UB

IAD

1(p

mo

l/m

g p

rote

in/m

in

UBIAD1-GFPTransmitted ER-Tracker Red Merge

50 µm

UBIAD1-GFPTransmittedBODIPY-TR

ceramide Merge

Subcellular localization of UBIAD1 in MG-63 cells

50 µm

UBIAD1-GFPTransmitted

MG-63 cells stably transfected with a UBIAD1–GFP expression vector were stained with ER-tracker Red or

BODIPY-TR ceramide (red) and were detected by GFP fluorescence (green). Merged images of GFP

fluorescence and by ER-marker or Golgi-marker fluorescence are shown at the right. The control construct

(mock-GFP) showed a diffuse fluorescence throughout the cytoplasm.

Nakagawa K. et al., Nature 2010; 468:117-121.

actin

)

1.2

1.4

1.6

1.8

1400

1600

1800

2000

MK-4

MK-4 epoxide

500

600

700

(pm

ol/g組織

)

MK-4-d7

MK-4-d7epoxide

UBIAD1 mRNA MK-4-d7 biosynthetic activity

Amounts of MK-4 and its epoxide

UBIAD1 mRNA expression, MK-4 biosynthetic activity, concentrations of MK-4 and its epoxide

in tissues of female mice

相対比

(UB

IAD

1/β

-actin

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

200

400

600

800

1000

1200

濃度

(pmol/g組織)

epoxide

0

100

200

300

400

K3-d

8からの生成量

(pm

ol/g

PK

MK-4

Prenylation

UBIAD1

Side-chain cleavage

GGPP

MK-4

R =

R=

O

O 3

3

OP

O

OH

OP

O

OH

HO

O

O

R

3

UBIAD1 is a novel biosynthetic enzyme for MK-4 that may

have both side-chain cleavage and prenylation activities

MK-4

K3

Side-chain cleavage

R= H3

Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme

Kimie Nakagawa, Yoshihisa Hirota, Natsumi Sawada, Naohito Yuge, Masato Watanabe, Yuri Uchino, Naoko Okuda, Yuka Shimomura, Yoshitomo Suhara & Toshio Okano

Department of Hygienic Sciences, Kobe Pharmaceutical University,

Nature 2010; 468:117-121.

O

O

D

D

D

CD3

D

D

K1-d7 K3-d8

Vitamin K homologues

O

O

D

D

D

CD3

D3

Mevalonic acid

Acetyl-CoA

Mevalonate pathway

HMG-CoA

HMG-CoA reductase

Statin

Bisphosphonate

MK-4 biosynthesis in tissues is decreased by the

treatments with statins and bisphosphonates

MK-4-d7

O

O

D

D

D

CD3

D3

UBIAD1

Geranylgeranyl pyrophosphate(GGPP)

Geranyl pyrophosphate(GPP)

Farnesyl pyrophosphate(FPP)

FPP synthase

PP

3

POO-

O

O O-

O-

O

OO

O

PO-

O-

O

P :P

phylloquinone

geranylgeranyl pyrophosphate

MK-4 Vitamin K cycle

MK-4（quinone form）

MK-4（hydroquinone

prenylation

Mevalonate pathway

Possible interaction of MK-4 biosynthesis, vitamin K cycle and vitamin K action

MK-4 synthetic Enz.

(UBIAD1)

SXR/PXR signaling

Biological functions

phytol

PKA/PKC signaling

VKDP（active form）

cycle

VKDP（inactive form）

GGCX CO2, O2MK-4-(epoxide

form)

（hydroquinone from）cleavage

Generation of Ubiad1 knockout miceA

Targeting vector

3.5 kb 1.1 kb 6.7 kb

1.7 kb

: loxP

: FRT

Ubiad1exon2

Ubiad1exon1

mTOR

exon1Wild-type allele

neoR

Cre recombinase

neoRDTA

SalI ClaII

NheI

SpeI

NotISacII

KpnI

Ubiad1-neofloxed allele

ATG

15001000

500300

(bp)

M.W.

+/− +/+ −/−Posi500 bp 100 bpB

Targeted allele

F RPCR primer

(A) Schematic presentation of ubiad1 genome, targeting

vector and disrupted Ubiad1 genome. (B) PCR genotyping of

Ubiad1+/+, Ubiad1+/− and Ubiad1−/− embryos. PCR

genotyping of tail DNA of Ubiad1+/+, Ubiad1+/− and

Ubiad1−/− embryos. Lane 1, positive controls for Ubiad1+/−

allele. Lane 2, PCR bands of Ubiad1+/− embryos. Lane 3,

PCR bands of Ubiad1+/+ embryos. Lane 4, PCR bands of

Ubiad1−/− embryos.

Nakagawa K, Okano T, et al, PLOS ONE 2014; 9: 1-12, e104078

Morphological examination of Ubiad1+/+, Ubiad1+/− and Ubiad1−/− embryos and weanling mice (postnatal day 1) from pregnant Ubiad1+/− mice orally administered CoQ10

6 ）））） Liver15

Brain

E

Day 1

DUbiad1+/+ Ubiad1+/− Ubiad1−/−Ubiad1+/+ Ubiad1+/− Ubiad1−/−

E15.5

HEstaining

A

B

0

2

4

Rela

tiv

e r

ati

o

(Ub

iad

1/G

ap

dh

）））） Liver

+/++/++/++/+ +/+/+/+/−−−− −−−−////−−−−0

5

10

Re

lati

ve

ra

tio

(Ub

iad

1/G

ap

dh

) Brain

N.D.N.D.

+/++/++/++/+ +/+/+/+/−−−− −−−−////−−−−

*

*

GAPDH

UBIAD1

F Ubiad1+/++/++/++/+ Ubiad1+/+/+/+/−−−− Ubiad1−−−−////−−−−

staining

C

UBIAD1staining

Ubiad1-deficient mouse embryos failed to survive beyond embryonic day 7.5 exhibiting small-sized body and gasturation arrest !!!

Flox/+ Cre/+

+/++/+

Cre/+

Flox+/+

+/+

Flox/+

Cre/+

-/+

Cre/+

Cre

Cre Cre

××××

Nestin-cre

Generation of a neural cell specific Ubiad1-/- mouse

flox/- flox/+cre/+

flox/+flox/-cre/+

Flox/+ Flox/+

+/+ Flox/Flox Flox/+ Flox/+

××××

Impairment of brain function

Co-Workers(Kobe Pharmaceutical University)

Kimie Nakagawa, Yoshihisa Hirota, Natsumi Sawada, Yuri Uchino, Masato Watanabe,

Syusaku Kimura, Naoto Yuge, Naoko Okuda, Yuka Shimomura, Makiko YamaneYuka Shimomura, Makiko Yamane

Dept. Hygienic SciencesKobe Pharmaceutical Univ.

(Shibaura Institute of Technology)Yoshitomo Suhara

Thank you for your attention!