Key nutrients in the

management of synaptic

failure a result of Alzheimer’s

disease

Patrick Kamphuis, PhD Global Head of Medical and Scientific Affairs

Nutricia, Schiphol, The Netherlands

Disclosure statement

• Employee of Nutricia Research

• No other relevant disclosures

Alzheimer’s disease: multiple pathologies, multiple approaches

AD

Amyloid Plaques

Synapse loss

Neurotransmitter deficits

Neurofibrillary tangles

T A R G E T

Synapse loss

Axon

neurite

dendritic spine

Sa

tura

ted

fa

tty a

cid

Glycerol

Phosphate

Choline

Synapses are built from neuronal membranes

Opportunity for a dietary approach

The Kennedy pathway for biosynthesis of neuronal membrane

Synapses consist principally of neuronal membranes

Axon

neurite

dendritic spine

Neurite

Dendritic spine

Axon terminal

Kennedy & Weiss (1956) J. Bio. Chem. 222, 193-214

Nutritional precursors enhance the synthesis rate of neuronal membranes

The Kennedy pathway for biosynthesis of neuronal membrane

Synapses consist principally of neuronal membranes

Kennedy & Weiss (1956) J. Bio. Chem. 222, 193-214

Nutritional precursors enhance the synthesis rate of neuronal membranes

Lower nutrient intake due to poor food

choices in early AD

DHA

vitamin B-6

vitamin B-12

Folate

vitamin C

vitamin E

Shatenstein et al., J Am Diet Assoc, 2007

Lower intake from food sources of

Phillips et al., Nutr Neurosci, 2012; Hart et al., Nutr Soc, 2013

Lower intake of

Fruits

Nuts

Seeds

Fish Higher intake of

Sugars & Snacks

• Deficiencies in essential nutrients can result in neurological

symptoms

• Diet and nutrient intake may change with age

• In AD patients, poor nutrition may be related to failure to remember

to eat, changes in eating preferences, and/or olfactory changes

• Epidemiological evidence suggests potential relationship between

nutrient intake and dementia – B-Vitamins

– Antioxidants – vitamins C and E

– Dietary fats

– Dietary patterns – i.e., Mediterranean diet

“ Nutrition and the brain “

link with Alzheimer’s disease (AD) ?

– Important nutrients

involved in synaptic

membrane synthesis

Nutrients and risk of AD

Engelhart et al., JAMA 2002

Collaboration in “Dementia’ project

Vitamin C intake

<95 96-133 >133 mg/d 0

1

Rela

tive R

isk

Rotterdam study

Barberger-Gateau et al., BMJ, 2002

Fish consumption

0

1

2

3

4

5

6

7

daily > weekly < weekly never

Incid

ence

(per

100 p

ers

ons

year)

AD Dementia

Dangour, Vellas et al., J Alz Dis 2010. Systematic review on Bvits and Fatty acids

Nutrient Author Journal #Subjects/

Duration Outcome

n3 PUFAs

Quinn

2010 JAMA

402

18 months

DHA compared with placebo did not slow the rate of cognitive and

functional decline in mild-moderate AD patients.

Freund-Levi

2006 Arch Neurol

174

6 months

No delay the rate of cognitive decline. However, positive effects were

observed in a small group of patients with very mild AD (MMSE>27)

Smith

2010 Plos

271

24 months

Accelerated rate of brain atrophy in mild cognitive impairment can be slowed by treatment with homocysteine-lowering B vitamins. No cognitive benefit. Post hoc (Jeneren 2015) links reduced atrophy to high DHA/EPA sub group.

B-vitamins Aisen

2008 JAMA

409

18 months

This regimen of high-dose B vitamin supplements does

not slow cognitive decline in individuals with mild to moderate AD.

McMahon

2006 N Eng J Med

276

24 months

The results of this trial do not support the hypothesis that homocysteine

lowering with B vitamins improves cognitive performance.

Vitamin E /

Antioxidants

Dysken

2014 JAMA

304

Mean f-up

27 months

Among patients with mild to moderate AD, 2000 IU/d of alpha-tocopherol

compared with placebo resulted in slower functional decline.

Petersen

2005 N Eng J Med

769

36 months Vitamin E had no benefit in patients with mild cognitive impairment.

Galasko

2012 Arch Neurol

52

16 weeks

However, this treatment (vitamin E + vitamin C plus α-lipoic acid) raised the

caution of faster cognitive decline

Vitamin D2 Stein

2011 J Alz Disease

32

8 weeks

We conclude that high-dose vitamin D provides

no benefit for cognition or disability over low-dose vitamin D

in mild-moderate AD

Ginkgo

biloba

DeKosky

2008 JAMA

3069

median f-up 6.1a

Ginkgo biloba at 120 mg twice a day was not effective in reducing either the

overall incidence rate of dementia or AD incidence in elderly individuals

with normal cognition or those with MCI.

But single nutrient intervention doesn‘t benefit

AD

MCI

MMSE

Psaltopoulou et al, Ann Neurol 2013

High adherence to Mediterranean diet reduces

risk of cognitive impairment

• Epidemiological evidence suggests certain dietary patterns can

reduce AD risk:

– Mediterranean Diet1,2

• Based on higher intakes of fish, vegetables, fruit

and monounsaturated fats

– DASH diet 3 • As above with emphasis on reduced intake

of dairy and salt

– MIND / Nordic / Finnish diet 4,5 • As per Mediterranean diet but with less fish and

fruit and more green vegetables and berries

1. Scarmeas N, et al . Mediterraneandiet and risk for Alzheimer’s disease. AnnNeurol 2006;59:912–21.

2. Martinez-Lapiscina EH, et al. Mediterranean diet improves cognition: the PREDIMED-NAVARRA randomised trial. J Neurol Neurosurg

Psychiatry 2013;84:1318–25

3 Tangney CC, et al. Relation of DASH- and Mediterranean-like dietary patterns on cognitive decline in older persons. Neurology

2014;83:1410–6.

4 Morris MC, et al. MIND diet associated with reduced incidence of Alzheimer’s disease. Alzheimer’s & Dementia 11 (2015) 1007-1014

5 Akesson A, et al. Health effects associated with foods characteristic of the Nordic diet. Food Nutr. Res57 ( 2013), 10.3402.

Epidemiological evidence suggests dietary

patterns can reduce AD risk

Systematic review & meta-analysis: nutrient

availability in AD

• According to Preferred Reporting Items for Systematic Reveiws and

Meta-Analysis (PRISMA) guidelines

• Analyses by independent statistics

% Cognitive Intact elderly

50

80

70

60

90

11

stu

die

s

10

stu

die

s

* *

6 s

tud

ies

9 s

tud

ies

*** *** *** ***

37

stu

die

s

31

stu

die

s

20

stu

die

s

8 s

tud

ies

11

stu

die

s

*

3 s

tud

ies

*

Meta-analyses De Wilde et al., poster AAIC 2014

Meta-analyses Lopes da Silva (2013) Alz Dement

*1,2,3

1:Trushina (2013) PLOS

2:Olde Rikkert (2014) JAD

3: Wang (2014) J Prot Res

Systematic review and meta-analysis of literature

Lower blood status in AD of nutrients

essential for synaptic membrane synthesis

*2

7 s

tud

ies

4 s

tud

ies

ns 3 % Cognitively Healthy elderly

50

80

70

60

90

110

*1 *1

*1 *1

*1 *1

12

stu

die

s

31

stu

die

s

4 s

tud

ies

9 s

tud

ies

5 s

tud

ies

5 s

tud

ies

ns

12

stu

die

s

ns

12

stu

die

s

1

1

De Wilde, AAIC 2016

Brain nutrient levels in AD versus cognitively healthy elderly

Lower brain status in AD of nutrients

essential for synaptic membrane synthesis

Increased nutritional need in AD

Lower nutrients levels in AD?

• Lower intake , and compromised endogenous synthesis

• Lower levels in blood

• Lower levels in the brain

Higher nutrient need due to synaptic membrane loss?

• Decreased synapse number in AD

• Nutrient control of membrane and synapse formation

Increased nutritional need cannot be met by the regular diet

Development of a Medical Food containing

Dietary precursors and cofactors

Stimulating synapse membrane formation requires specific nutrients

Lower Nutrient status

Uridine (UMP), Omega-3 fatty acids, Phosholipids & Choline, B-Vitamins, Antioxidants

Development of a Medical Food containing

Dietary precursors and cofactors

400 mg Choline 300 mg EPA

625 mg UMP

3 mcg Vit B12

1 mg Vit B6

80 mg Vit C

1200 mg DHA

400 mcg Folate

106 mg Phospholipids

60 mcg Selenium 40 mg Vit E

Increased nutritional need cannot be met by the regular diet

Development of a Medical Food containing

Dietary precursors and cofactors

Stimulating synapse membrane formation requires specific nutrients

Lower Nutrient status

HYPOTHESIS: Souvenaid successfully addresses an unmet nutritional need in people with AD by

increasing their intake of these dietary precursors and co-factors

Uridine (UMP), Omega-3 fatty acids, Phosholipids & Choline, B-Vitamins, Antioxidants

Improved memory in mild and very mild AD

0

10

20

30

40

50

worsened unchanged improved

% o

f p

atie

nts

Control Souvenaid

0

10

20

30

40

50

worsened unchanged improved

2008 - FIRST HUMAN TRIAL SHOWED THAT

SOUVENAID IMPROVED MEMORY SCORES IN

PEOPLE WITH MILD AD

The Souvenaid clinical trial program

aMCI due to AD Moderate AD

Souvenir II

Souvenir I

S-Connect

Souvenir II

Open Label

NTB + EEG

n=259

NL, Ger, Bel, Fr, It, Sp

WMS-r & ADAS-cog

n=225

NL, Ger, Bel, UK, US

Safety + Compliance

+ NTB

Mild AD

This project receives funding from the NL Food & Nutrition Delta project, FND N°10003

This project receives funding from NL STW

This project receives funding from the NL Food & Nutrition Delta project, FND N°10003

ADAS-cog

n=527 (MMSE 14-24); stable on AD drugs; US

com

ple

ted &

publish

ed

Souvenir II: design and methodology

• Multi-country randomized, controlled trial in drug naive mild AD

over 24 weeks, with a further 24 weeks open label phase

• Primary outcome: Memory Domain NTB (z-score) at 24 weeks

• RAVLT immediate, delayed, recognition and VPA immediate and

delayed

n=259 Souvenaid (n=130)

0 12 24 48

Control (n=129)

Outcome parameters

Values are mean±SD, unless stated otherwise

Baseline Characteristics Control

(n = 129) Souvenaid (n = 130)

Age (y) 73.2 (8.4) 74.4 (6.9)

Sex: males (n[%]) 64 (49.6) 68 (52.3)

Years of education on top of primary school

6.6 (4.6) 6.5 (4.8)

Total MMSE score 25.1 (2.9) 25.1 (2.8)

Duration AD since diagnosis (months) (median[range])

2.0 (0.0 - 88.0) 1.0 (0.0 - 70.0)

BMI (kg/m2) 26.7 (4.2) 26.1 (4.1)

Souvenaid

Souvenaid

Double blind phase Open Label

Sustained improvement in memory performance over 24 and 48 weeks

Seconday: NTB composite z-score trend (p=0.053)

Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36

24 wk vs baseline p = 0.035

Primary: memory domain z-score (p=0.023)

24 wk vs baseline p = 0.090 24 wk vs baseline p = 0.035

Sustained improvement in memory performance over 24 and 48 weeks

Seconday: NTB composite z-score trend (p=0.053)

Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36

24 wk vs baseline p = 0.035

Primary: memory domain z-score (p=0.023)

24 wk vs baseline p = 0.090 24 wk vs baseline p = 0.035

Active - Active: p=0.038

Control - Active: p=0.029

Olde Rikkert et al. J Alzheimers Dis. 2015

Souvenaid increases nutrient blood levels -

Data from Souvenir II & Open label extension

Folate

B12

B6

Hcy

Choline

Uridine

eDHA

eEPA Selenium

Vit E

pla DHA

pla EPA

0 24

0

35

45

DH

A

(% t

ota

l fat

ty a

cid

s er

yth

rocy

te m

emb

ran

e)

48

Control Souvenaid

Souvenaid

Double-blind phase Open-label phase 30

40

Sample size (N)

128 129

119 114

93 87

Time (weeks)

Souvenaid increases nutrient blood levels -

Data from Souvenir II & Open label extension

Folate

B12

B6

Hcy

Choline

Uridine

eDHA

eEPA Selenium

Vit E

pla DHA

pla EPA

Level 1: basic signal analysis

Level 2: connectivity analysis

Level 3: network analysis

In AD: slowing of the oscillations

In AD: decreased functional connectivity

In AD: disruption of optimal organization

Snaedal et al. 2010

Babiloni et al. 2004

Stam 2004

Souvenaid increases EEG biomarkers for functional connectivity, derivatives of synaptic activity

De Waal et al. , PlosOne, 2014; Scheltens et al., J Alz Dis, 2012

Level 1: basic signal analysis

Level 2: connectivity analysis

Level 3: network analysis

In AD: slowing of the oscillations

In AD: decreased functional connectivity

In AD: disruption of optimal organization

Snaedal et al. 2010

Babiloni et al. 2004

Stam 2004

Souvenaid increases EEG biomarkers for functional connectivity, derivatives of synaptic activity

De Waal et al. , PlosOne, 2014; Scheltens et al., J Alz Dis, 2012

p = 0.019

p = 0.011

p = 0.009 p = 0.053

Level 1: basic signal analysis

Level 2: connectivity analysis

Level 3: network analysis

In AD: slowing of the oscillations

In AD: decreased functional connectivity

In AD: disruption of optimal organization

Snaedal et al. 2010

Babiloni et al. 2004

Stam 2004

Souvenaid increases EEG biomarkers for functional connectivity, derivatives of synaptic activity

De Waal et al. , PlosOne, 2014; Scheltens et al., J Alz Dis, 2012

p = 0.019

p = 0.011

p = 0.009 p = 0.053

Lower levels of multiple key nutrients

in early AD (MMSE 25) compared to age-matched

healthy controls

P = 0.046

No significant differences B12 (p=0.158), Folate (p=0.309)

Olde-Rikkert et al., J Alz Dis, accepted

• Baseline data from 79 AD

patients

• Data collected at single time-

point for 93 healthy subjects

matched for age, gender and

country

Lower levels of multiple key nutrients

in early AD (MMSE 25) compared to age-matched

healthy controls

P = 0.046

No significant differences B12 (p=0.158), Folate (p=0.309)

Olde-Rikkert et al., J Alz Dis, accepted

• Baseline data from 79 AD

patients

• Data collected at single time-

point for 93 healthy subjects

matched for age, gender and

country

MRS Study: Design & methodology

• Goal: A Magnetic Resonance Spectroscopy (MRS) study to

explore the effects of Souvenaid on brain phospholipid

metabolites in patients with mild Alzheimer’s disease

dementia (AD)

• Experimental set up: 4-week, randomised, controlled,

double blind, parallel-group, single-centre, exploratory study

in 33 drug naïve mild AD patients

Rijpma et al. Neurobiol Aging 2016 39 (S1) S7-8

The Souvenaid clinical trial program

aMCI due to AD Moderate AD

NTB + MRI / CSF n=311(MMSE ≥ 24); Ger, NL, FI, SW

LipiDiDiet

MRS study

Souvenir II

Souvenir I

S-Connect

Open Label Safety + Compliance

+ NTB

31P and 1H-MRS 30 drug-naïve, NL

Mild AD

FDG-PET FDG-PET 40 drug-naïve, NL

Funded by the EU FP7 project LipiDiDiet, Grant Agreement N° 211696

This project receives funding from the NL Food & Nutrition Delta project, FND N°10003

This project receives funding from NL STW

This project receives funding by the NL NWO-FCB

This project receives funding from the NL Food & Nutrition Delta project, FND N°10003

MIND-AD Feasibility, tbd This project is funding by the Joint Programming Neurodegenerative Disease Research

ADAS-cog

n=527 (MMSE 14-24); stable on AD drugs; US

WMS-r & ADAS-cog

n=225

NL, Ger, Bel, UK, US

NTB + EEG

n=259

NL, Ger, Bel, Fr, It, Sp

Favorable safety profile across clinical trial

program – Souvenaid is well tolerated

Trial Total trial n=

Total AEs Control AEs

Active AEs

P-value

SI 225 107 49 58 ns

SII 259 239 135 104 ns

S-Connect 527 903 445 458 ns

SII open label 201 135 135 -

LDD 311 270 138 132 ns

High compliance across all the trials: >95%

NNH values for Souvenaid are very high or not calculable, in accordance with

its good safety and tolerability profile

• AE’s are consistent with that expected for elderly population with AD

• No SAE’s considered to be related to the study product

Lower vitamin, mineral and omega/3 fatty

acid nutrient status in AD: Meta-analyses

Similar results for plasma levels of vitamins C and E,

& DHA

Unadjusted Overall (REML, I-squared=87%, P=0.001)

Kristensen '93

Morillas-Ruiz '10

Age adjusted Overall (REML meta-regression, P<0.001)

Joosten '97

Basun '94

Clarke '98

Religa '03

Lelhuber '00

Ravaglia '00

study

Regland '92

Karimi '09

Parnetti '92

Dominguez '05

Nagga '03

O'Neill '90

Irizarry '05`"'

Mizrahi '04

Linnebank '10

Villa '09

Li '04

Anello '04

Basun '94

Nilsson '04

Postiglione '01

Hogervorst '02

Ravaglia '04

Malaguarnera '04

Glaso '04

Cascalheira '09

Selley '02

Asita De Silva '05

Koseoglu '07

Basun '94

Quadri '05

Galluci '04

Basun '94

Basun '94

Faux '11

20

48

49

117

108

100

19

13

control

32

49

26

19

101

9

88

155

60

18

30

181

6

51

74

62

29

30

18

36

25

21

40

58

79

n

42

112

99

760

256

441

210

290

253

305

283

327

control

261

269

260

713

307

290

390

351

248

369

314

283

198

257

780

346

308

437

435

345

322

365

288

239

275

mean

354

249

305

300

103

217

91

137

100

178

139

69

control

110

211

117

256

153

93

179

175

103

199

184

211

41

129

211

116

127

58

263

26

68

103

15

116

96

sd

297

117

128

132

26

52

52

26

164

99

19

34

AD

53

51

52

29

47

9

145

75

60

20

30

180

3

159

74

66

51

30

20

19

27

23

51

15

111

n

137

28

22

205

179

454

210

294

236

234

260

282

AD

251

288

262

468

274

313

429

323

272

334

217

278

279

254

491

261

292

370

350

198

254

357

207

319

272

mean

277

259

246

299

92

300

150

169

112

103

95

24

AD

93

182

150

218

80

82

220

136

192

126

134

221

91

97

144

108

96

77

264

94

77

99

15

143

108

sd

169

105

103

139

-35.83 (-57.30, -14.36)

-77.00 (-134.24, -19.76)

13.01 (-89.05, 115.07)

-47.88 (-70.75, -25.01)

0.00 (-48.08, 48.08)

4.00 (-65.54, 73.54)

-17.00 (-42.49, 8.49)

-71.43 (-111.80, -31.06)

-22.81 (-98.58, 52.96)

-45.00 (-83.50, -6.50)

mean difference (95% CI)

-10.42 (-56.02, 35.18)

19.19 (-58.11, 96.49)

1.93 (-58.64, 62.50)

-245.35 (-385.21, -105.49)

-33.00 (-70.60, 4.60)

22.88 (-58.08, 103.84)

39.00 (-12.78, 90.78)

-27.60 (-68.94, 13.74)

24.00 (-31.13, 79.13)

-35.80 (-142.91, 71.31)

-96.30 (-177.82, -14.78)

-5.00 (-49.58, 39.58)

81.00 (-27.07, 189.07)

-3.00 (-41.48, 35.48)

-289.00 (-347.20, -230.80)

-85.00 (-123.89, -46.11)

-16.00 (-69.20, 37.20)

-67.20 (-101.74, -32.66)

-85.00 (-252.77, 82.77)

-146.87 (-190.18, -103.56)

-68.80 (-108.19, -29.41)

-8.10 (-67.86, 51.66)

-80.52 (-86.84, -74.20)

80.00 (1.72, 158.28)

-3.00 (-32.19, 26.19)

-76.62 (-170.85, 17.61)

10.00 (-34.52, 54.52)

-59.00 (-108.88, -9.12)

-1.40 (-22.61, 19.81)

2.8

1.9

2.9

2.5

3.3

3.1

2.4

3.1

(%)

3.0

2.4

2.7

1.4

3.1

2.3

2.9

3.1

2.8

1.9

2.3

3.0

1.8

3.1

2.8

3.1

2.8

3.2

1.1

3.0

3.1

2.7

3.4

2.4

3.2

weight

2.1

3.0

2.9

3.3

-35.83 (-57.30, -14.36)

-77.00 (-134.24, -19.76)

13.01 (-89.05, 115.07)

-47.88 (-70.75, -25.01)

0.00 (-48.08, 48.08)

4.00 (-65.54, 73.54)

-17.00 (-42.49, 8.49)

-71.43 (-111.80, -31.06)

-22.81 (-98.58, 52.96)

-45.00 (-83.50, -6.50)

mean difference (95% CI)

-10.42 (-56.02, 35.18)

19.19 (-58.11, 96.49)

1.93 (-58.64, 62.50)

-245.35 (-385.21, -105.49)

-33.00 (-70.60, 4.60)

22.88 (-58.08, 103.84)

39.00 (-12.78, 90.78)

-27.60 (-68.94, 13.74)

24.00 (-31.13, 79.13)

-35.80 (-142.91, 71.31)

-96.30 (-177.82, -14.78)

-5.00 (-49.58, 39.58)

81.00 (-27.07, 189.07)

-3.00 (-41.48, 35.48)

-289.00 (-347.20, -230.80)

-85.00 (-123.89, -46.11)

-16.00 (-69.20, 37.20)

-67.20 (-101.74, -32.66)

-85.00 (-252.77, 82.77)

-146.87 (-190.18, -103.56)

-68.80 (-108.19, -29.41)

-8.10 (-67.86, 51.66)

-80.52 (-86.84, -74.20)

80.00 (1.72, 158.28)

-3.00 (-32.19, 26.19)

-76.62 (-170.85, 17.61)

10.00 (-34.52, 54.52)

-59.00 (-108.88, -9.12)

-1.40 (-22.61, 19.81)

2.8

1.9

2.9

2.5

3.3

3.1

2.4

3.1

(%)

3.0

2.4

2.7

1.4

3.1

2.3

2.9

3.1

2.8

1.9

2.3

3.0

1.8

3.1

2.8

3.1

2.8

3.2

1.1

3.0

3.1

2.7

3.4

2.4

3.2

weight

2.1

3.0

2.9

3.3

AD lower AD higher

0-400 -300 -200 -100 0 100 200

vitamin B12

Unadjusted Overall (REML, I-squared=87%, P=0.001)

Kristensen '93

Morillas-Ruiz '10

Age adjusted Overall (REML meta-regression, P<0.001)

Joosten '97

Basun '94

Clarke '98

Religa '03

Lelhuber '00

Ravaglia '00

study

Regland '92

Karimi '09

Parnetti '92

Dominguez '05

Nagga '03

O'Neill '90

Irizarry '05`"'

Mizrahi '04

Linnebank '10

Villa '09

Li '04

Anello '04

Basun '94

Nilsson '04

Postiglione '01

Hogervorst '02

Ravaglia '04

Malaguarnera '04

Glaso '04

Cascalheira '09

Selley '02

Asita De Silva '05

Koseoglu '07

Basun '94

Quadri '05

Galluci '04

Basun '94

Basun '94

Faux '11

20

48

49

117

108

100

19

13

control

32

49

26

19

101

9

88

155

60

18

30

181

6

51

74

62

29

30

18

36

25

21

40

58

79

n

42

112

99

760

256

441

210

290

253

305

283

327

control

261

269

260

713

307

290

390

351

248

369

314

283

198

257

780

346

308

437

435

345

322

365

288

239

275

mean

354

249

305

300

103

217

91

137

100

178

139

69

control

110

211

117

256

153

93

179

175

103

199

184

211

41

129

211

116

127

58

263

26

68

103

15

116

96

sd

297

117

128

132

26

52

52

26

164

99

19

34

AD

53

51

52

29

47

9

145

75

60

20

30

180

3

159

74

66

51

30

20

19

27

23

51

15

111

n

137

28

22

205

179

454

210

294

236

234

260

282

AD

251

288

262

468

274

313

429

323

272

334

217

278

279

254

491

261

292

370

350

198

254

357

207

319

272

mean

277

259

246

299

92

300

150

169

112

103

95

24

AD

93

182

150

218

80

82

220

136

192

126

134

221

91

97

144

108

96

77

264

94

77

99

15

143

108

sd

169

105

103

139

-35.83 (-57.30, -14.36)

-77.00 (-134.24, -19.76)

13.01 (-89.05, 115.07)

-47.88 (-70.75, -25.01)

0.00 (-48.08, 48.08)

4.00 (-65.54, 73.54)

-17.00 (-42.49, 8.49)

-71.43 (-111.80, -31.06)

-22.81 (-98.58, 52.96)

-45.00 (-83.50, -6.50)

mean difference (95% CI)

-10.42 (-56.02, 35.18)

19.19 (-58.11, 96.49)

1.93 (-58.64, 62.50)

-245.35 (-385.21, -105.49)

-33.00 (-70.60, 4.60)

22.88 (-58.08, 103.84)

39.00 (-12.78, 90.78)

-27.60 (-68.94, 13.74)

24.00 (-31.13, 79.13)

-35.80 (-142.91, 71.31)

-96.30 (-177.82, -14.78)

-5.00 (-49.58, 39.58)

81.00 (-27.07, 189.07)

-3.00 (-41.48, 35.48)

-289.00 (-347.20, -230.80)

-85.00 (-123.89, -46.11)

-16.00 (-69.20, 37.20)

-67.20 (-101.74, -32.66)

-85.00 (-252.77, 82.77)

-146.87 (-190.18, -103.56)

-68.80 (-108.19, -29.41)

-8.10 (-67.86, 51.66)

-80.52 (-86.84, -74.20)

80.00 (1.72, 158.28)

-3.00 (-32.19, 26.19)

-76.62 (-170.85, 17.61)

10.00 (-34.52, 54.52)

-59.00 (-108.88, -9.12)

-1.40 (-22.61, 19.81)

2.8

1.9

2.9

2.5

3.3

3.1

2.4

3.1

(%)

3.0

2.4

2.7

1.4

3.1

2.3

2.9

3.1

2.8

1.9

2.3

3.0

1.8

3.1

2.8

3.1

2.8

3.2

1.1

3.0

3.1

2.7

3.4

2.4

3.2

weight

2.1

3.0

2.9

3.3

-35.83 (-57.30, -14.36)

-77.00 (-134.24, -19.76)

13.01 (-89.05, 115.07)

-47.88 (-70.75, -25.01)

0.00 (-48.08, 48.08)

4.00 (-65.54, 73.54)

-17.00 (-42.49, 8.49)

-71.43 (-111.80, -31.06)

-22.81 (-98.58, 52.96)

-45.00 (-83.50, -6.50)

mean difference (95% CI)

-10.42 (-56.02, 35.18)

19.19 (-58.11, 96.49)

1.93 (-58.64, 62.50)

-245.35 (-385.21, -105.49)

-33.00 (-70.60, 4.60)

22.88 (-58.08, 103.84)

39.00 (-12.78, 90.78)

-27.60 (-68.94, 13.74)

24.00 (-31.13, 79.13)

-35.80 (-142.91, 71.31)

-96.30 (-177.82, -14.78)

-5.00 (-49.58, 39.58)

81.00 (-27.07, 189.07)

-3.00 (-41.48, 35.48)

-289.00 (-347.20, -230.80)

-85.00 (-123.89, -46.11)

-16.00 (-69.20, 37.20)

-67.20 (-101.74, -32.66)

-85.00 (-252.77, 82.77)

-146.87 (-190.18, -103.56)

-68.80 (-108.19, -29.41)

-8.10 (-67.86, 51.66)

-80.52 (-86.84, -74.20)

80.00 (1.72, 158.28)

-3.00 (-32.19, 26.19)

-76.62 (-170.85, 17.61)

10.00 (-34.52, 54.52)

-59.00 (-108.88, -9.12)

-1.40 (-22.61, 19.81)

2.8

1.9

2.9

2.5

3.3

3.1

2.4

3.1

(%)

3.0

2.4

2.7

1.4

3.1

2.3

2.9

3.1

2.8

1.9

2.3

3.0

1.8

3.1

2.8

3.1

2.8

3.2

1.1

3.0

3.1

2.7

3.4

2.4

3.2

weight

2.1

3.0

2.9

3.3

AD lower AD higher

0-400 -300 -200 -100 0 100 200

vitamin B12

Unadjusted Overall (REML, I-squared=87%, P=0.001)

Kristensen '93

Morillas-Ruiz '10

Age adjusted Overall (REML meta-regression, P<0.001)

Joosten '97

Basun '94

Clarke '98

Religa '03

Lelhuber '00

Ravaglia '00

study

Regland '92

Karimi '09

Parnetti '92

Dominguez '05

Nagga '03

O'Neill '90

Irizarry '05`"'

Mizrahi '04

Linnebank '10

Villa '09

Li '04

Anello '04

Basun '94

Nilsson '04

Postiglione '01

Hogervorst '02

Ravaglia '04

Malaguarnera '04

Glaso '04

Cascalheira '09

Selley '02

Asita De Silva '05

Koseoglu '07

Basun '94

Quadri '05

Galluci '04

Basun '94

Basun '94

Faux '11

20

48

49

117

108

100

19

13

control

32

49

26

19

101

9

88

155

60

18

30

181

6

51

74

62

29

30

18

36

25

21

40

58

79

n

42

112

99

760

256

441

210

290

253

305

283

327

control

261

269

260

713

307

290

390

351

248

369

314

283

198

257

780

346

308

437

435

345

322

365

288

239

275

mean

354

249

305

300

103

217

91

137

100

178

139

69

control

110

211

117

256

153

93

179

175

103

199

184

211

41

129

211

116

127

58

263

26

68

103

15

116

96

sd

297

117

128

132

26

52

52

26

164

99

19

34

AD

53

51

52

29

47

9

145

75

60

20

30

180

3

159

74

66

51

30

20

19

27

23

51

15

111

n

137

28

22

205

179

454

210

294

236

234

260

282

AD

251

288

262

468

274

313

429

323

272

334

217

278

279

254

491

261

292

370

350

198

254

357

207

319

272

mean

277

259

246

299

92

300

150

169

112

103

95

24

AD

93

182

150

218

80

82

220

136

192

126

134

221

91

97

144

108

96

77

264

94

77

99

15

143

108

sd

169

105

103

139

-35.83 (-57.30, -14.36)

-77.00 (-134.24, -19.76)

13.01 (-89.05, 115.07)

-47.88 (-70.75, -25.01)

0.00 (-48.08, 48.08)

4.00 (-65.54, 73.54)

-17.00 (-42.49, 8.49)

-71.43 (-111.80, -31.06)

-22.81 (-98.58, 52.96)

-45.00 (-83.50, -6.50)

mean difference (95% CI)

-10.42 (-56.02, 35.18)

19.19 (-58.11, 96.49)

1.93 (-58.64, 62.50)

-245.35 (-385.21, -105.49)

-33.00 (-70.60, 4.60)

22.88 (-58.08, 103.84)

39.00 (-12.78, 90.78)

-27.60 (-68.94, 13.74)

24.00 (-31.13, 79.13)

-35.80 (-142.91, 71.31)

-96.30 (-177.82, -14.78)

-5.00 (-49.58, 39.58)

81.00 (-27.07, 189.07)

-3.00 (-41.48, 35.48)

-289.00 (-347.20, -230.80)

-85.00 (-123.89, -46.11)

-16.00 (-69.20, 37.20)

-67.20 (-101.74, -32.66)

-85.00 (-252.77, 82.77)

-146.87 (-190.18, -103.56)

-68.80 (-108.19, -29.41)

-8.10 (-67.86, 51.66)

-80.52 (-86.84, -74.20)

80.00 (1.72, 158.28)

-3.00 (-32.19, 26.19)

-76.62 (-170.85, 17.61)

10.00 (-34.52, 54.52)

-59.00 (-108.88, -9.12)

-1.40 (-22.61, 19.81)

2.8

1.9

2.9

2.5

3.3

3.1

2.4

3.1

(%)

3.0

2.4

2.7

1.4

3.1

2.3

2.9

3.1

2.8

1.9

2.3

3.0

1.8

3.1

2.8

3.1

2.8

3.2

1.1

3.0

3.1

2.7

3.4

2.4

3.2

weight

2.1

3.0

2.9

3.3

-35.83 (-57.30, -14.36)

-77.00 (-134.24, -19.76)

13.01 (-89.05, 115.07)

-47.88 (-70.75, -25.01)

0.00 (-48.08, 48.08)

4.00 (-65.54, 73.54)

-17.00 (-42.49, 8.49)

-71.43 (-111.80, -31.06)

-22.81 (-98.58, 52.96)

-45.00 (-83.50, -6.50)

mean difference (95% CI)

-10.42 (-56.02, 35.18)

19.19 (-58.11, 96.49)

1.93 (-58.64, 62.50)

-245.35 (-385.21, -105.49)

-33.00 (-70.60, 4.60)

22.88 (-58.08, 103.84)

39.00 (-12.78, 90.78)

-27.60 (-68.94, 13.74)

24.00 (-31.13, 79.13)

-35.80 (-142.91, 71.31)

-96.30 (-177.82, -14.78)

-5.00 (-49.58, 39.58)

81.00 (-27.07, 189.07)

-3.00 (-41.48, 35.48)

-289.00 (-347.20, -230.80)

-85.00 (-123.89, -46.11)

-16.00 (-69.20, 37.20)

-67.20 (-101.74, -32.66)

-85.00 (-252.77, 82.77)

-146.87 (-190.18, -103.56)

-68.80 (-108.19, -29.41)

-8.10 (-67.86, 51.66)

-80.52 (-86.84, -74.20)

80.00 (1.72, 158.28)

-3.00 (-32.19, 26.19)

-76.62 (-170.85, 17.61)

10.00 (-34.52, 54.52)

-59.00 (-108.88, -9.12)

-1.40 (-22.61, 19.81)

2.8

1.9

2.9

2.5

3.3

3.1

2.4

3.1

(%)

3.0

2.4

2.7

1.4

3.1

2.3

2.9

3.1

2.8

1.9

2.3

3.0

1.8

3.1

2.8

3.1

2.8

3.2

1.1

3.0

3.1

2.7

3.4

2.4

3.2

weight

2.1

3.0

2.9

3.3

AD lower AD higher

0-400 -300 -200 -100 0 100 200

vitamin B12

Unadjusted Overall (REML, I-squared=88%, P<0.001)

Ravaglia '04

Morillas-Ruiz '10

Postiglione '01

Villa '09

Lelhuber '00

Cascalheira '09

Mizrahi '04

Selley '02

Karimi '09

Koseoglu '07

Galimberti '08

Lovati '07

Religa '03

Age adjusted Overall (REML meta-regression, P<0.001)

Serot '01

Faux '11

Regland '92

Malaguarnera '04

Dominguez '05

Clarke '98

Hogervorst '02

Quadri '05

study

Galluci '04

Anello '04

Li '04

Linnebank '10

Agarwal '10

Joosten '97

Asita De Silva '05

Irizarry '05

Parnetti '92

29

48

74

18

19

36

155

25

49

40

23

76

100

28

760

32

30

19

108

62

79

control

42

181

30

60

127

49

21

88

26

n

16.6

28.8

8.5

19.0

14.3

20.4

4.8

25.1

15.9

28.1

19.8

15.6

17.1

13.2

30.3

20.0

13.6

29.6

22.9

24.9

16.8

control

14.1

15.7

37.2

14.1

15.7

8.6

19.7

35.2

14.1

mean

7.3

7.7

3.2

4.1

9.3

1.7

2.6

4.7

8.6

3.4

6.2

7.9

12.2

4.8

12.7

18.0

3.2

9.0

10.0

11.3

5.5

control

11.1

5.9

21.2

7.7

30.1

3.2

9.7

32.9

1.1

sd

51

52

74

20

19

19

75

27

51

51

29

108

99

30

205

53

30

29

164

66

111

AD

137

180

30

60

32

52

23

145

52

n

11.1

21.8

5.7

16.8

10.0

18.8

4.3

14.7

14.5

21.4

8.6

8.2

19.3

12.1

29.4

16.7

10.6

17.9

17.6

15.9

13.1

AD

11.6

14.3

29.2

15.6

15.0

7.9

15.9

29.9

9.5

mean

4.3

8.7

2.1

4.7

3.4

5.3

3.2

4.3

6.6

4.4

2.8

5.3

7.7

4.9

14.5

15.5

3.2

7.2

10.7

11.3

5.9

AD

6.1

5.7

12.7

7.0

14.7

4.2

8.4

21.3

1.1

sd

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

AD lower AD higher

0-20 -15 -10 -5 0 5 10

folate

Unadjusted Overall (REML, I-squared=88%, P<0.001)

Ravaglia '04

Morillas-Ruiz '10

Postiglione '01

Villa '09

Lelhuber '00

Cascalheira '09

Mizrahi '04

Selley '02

Karimi '09

Koseoglu '07

Galimberti '08

Lovati '07

Religa '03

Age adjusted Overall (REML meta-regression, P<0.001)

Serot '01

Faux '11

Regland '92

Malaguarnera '04

Dominguez '05

Clarke '98

Hogervorst '02

Quadri '05

study

Galluci '04

Anello '04

Li '04

Linnebank '10

Agarwal '10

Joosten '97

Asita De Silva '05

Irizarry '05

Parnetti '92

29

48

74

18

19

36

155

25

49

40

23

76

100

28

760

32

30

19

108

62

79

control

42

181

30

60

127

49

21

88

26

n

16.6

28.8

8.5

19.0

14.3

20.4

4.8

25.1

15.9

28.1

19.8

15.6

17.1

13.2

30.3

20.0

13.6

29.6

22.9

24.9

16.8

control

14.1

15.7

37.2

14.1

15.7

8.6

19.7

35.2

14.1

mean

7.3

7.7

3.2

4.1

9.3

1.7

2.6

4.7

8.6

3.4

6.2

7.9

12.2

4.8

12.7

18.0

3.2

9.0

10.0

11.3

5.5

control

11.1

5.9

21.2

7.7

30.1

3.2

9.7

32.9

1.1

sd

51

52

74

20

19

19

75

27

51

51

29

108

99

30

205

53

30

29

164

66

111

AD

137

180

30

60

32

52

23

145

52

n

11.1

21.8

5.7

16.8

10.0

18.8

4.3

14.7

14.5

21.4

8.6

8.2

19.3

12.1

29.4

16.7

10.6

17.9

17.6

15.9

13.1

AD

11.6

14.3

29.2

15.6

15.0

7.9

15.9

29.9

9.5

mean

4.3

8.7

2.1

4.7

3.4

5.3

3.2

4.3

6.6

4.4

2.8

5.3

7.7

4.9

14.5

15.5

3.2

7.2

10.7

11.3

5.9

AD

6.1

5.7

12.7

7.0

14.7

4.2

8.4

21.3

1.1

sd

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

AD lower AD higher

0-20 -15 -10 -5 0 5 10

folate

Unadjusted Overall (REML, I-squared=88%, P<0.001)

Ravaglia '04

Morillas-Ruiz '10

Postiglione '01

Villa '09

Lelhuber '00

Cascalheira '09

Mizrahi '04

Selley '02

Karimi '09

Koseoglu '07

Galimberti '08

Lovati '07

Religa '03

Age adjusted Overall (REML meta-regression, P<0.001)

Serot '01

Faux '11

Regland '92

Malaguarnera '04

Dominguez '05

Clarke '98

Hogervorst '02

Quadri '05

study

Galluci '04

Anello '04

Li '04

Linnebank '10

Agarwal '10

Joosten '97

Asita De Silva '05

Irizarry '05

Parnetti '92

29

48

74

18

19

36

155

25

49

40

23

76

100

28

760

32

30

19

108

62

79

control

42

181

30

60

127

49

21

88

26

n

16.6

28.8

8.5

19.0

14.3

20.4

4.8

25.1

15.9

28.1

19.8

15.6

17.1

13.2

30.3

20.0

13.6

29.6

22.9

24.9

16.8

control

14.1

15.7

37.2

14.1

15.7

8.6

19.7

35.2

14.1

mean

7.3

7.7

3.2

4.1

9.3

1.7

2.6

4.7

8.6

3.4

6.2

7.9

12.2

4.8

12.7

18.0

3.2

9.0

10.0

11.3

5.5

control

11.1

5.9

21.2

7.7

30.1

3.2

9.7

32.9

1.1

sd

51

52

74

20

19

19

75

27

51

51

29

108

99

30

205

53

30

29

164

66

111

AD

137

180

30

60

32

52

23

145

52

n

11.1

21.8

5.7

16.8

10.0

18.8

4.3

14.7

14.5

21.4

8.6

8.2

19.3

12.1

29.4

16.7

10.6

17.9

17.6

15.9

13.1

AD

11.6

14.3

29.2

15.6

15.0

7.9

15.9

29.9

9.5

mean

4.3

8.7

2.1

4.7

3.4

5.3

3.2

4.3

6.6

4.4

2.8

5.3

7.7

4.9

14.5

15.5

3.2

7.2

10.7

11.3

5.9

AD

6.1

5.7

12.7

7.0

14.7

4.2

8.4

21.3

1.1

sd

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

AD lower AD higher

0-20 -15 -10 -5 0 5 10

folate

Unadjusted Overall (REML, I-squared=88%, P<0.001)

Ravaglia '04

Morillas-Ruiz '10

Postiglione '01

Villa '09

Lelhuber '00

Cascalheira '09

Mizrahi '04

Selley '02

Karimi '09

Koseoglu '07

Galimberti '08

Lovati '07

Religa '03

Age adjusted Overall (REML meta-regression, P<0.001)

Serot '01

Faux '11

Regland '92

Malaguarnera '04

Dominguez '05

Clarke '98

Hogervorst '02

Quadri '05

study

Galluci '04

Anello '04

Li '04

Linnebank '10

Agarwal '10

Joosten '97

Asita De Silva '05

Irizarry '05

Parnetti '92

29

48

74

18

19

36

155

25

49

40

23

76

100

28

760

32

30

19

108

62

79

control

42

181

30

60

127

49

21

88

26

n

16.6

28.8

8.5

19.0

14.3

20.4

4.8

25.1

15.9

28.1

19.8

15.6

17.1

13.2

30.3

20.0

13.6

29.6

22.9

24.9

16.8

control

14.1

15.7

37.2

14.1

15.7

8.6

19.7

35.2

14.1

mean

7.3

7.7

3.2

4.1

9.3

1.7

2.6

4.7

8.6

3.4

6.2

7.9

12.2

4.8

12.7

18.0

3.2

9.0

10.0

11.3

5.5

control

11.1

5.9

21.2

7.7

30.1

3.2

9.7

32.9

1.1

sd

51

52

74

20

19

19

75

27

51

51

29

108

99

30

205

53

30

29

164

66

111

AD

137

180

30

60

32

52

23

145

52

n

11.1

21.8

5.7

16.8

10.0

18.8

4.3

14.7

14.5

21.4

8.6

8.2

19.3

12.1

29.4

16.7

10.6

17.9

17.6

15.9

13.1

AD

11.6

14.3

29.2

15.6

15.0

7.9

15.9

29.9

9.5

mean

4.3

8.7

2.1

4.7

3.4

5.3

3.2

4.3

6.6

4.4

2.8

5.3

7.7

4.9

14.5

15.5

3.2

7.2

10.7

11.3

5.9

AD

6.1

5.7

12.7

7.0

14.7

4.2

8.4

21.3

1.1

sd

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

-3.88 (-5.13, -2.64)

-5.47 (-8.36, -2.58)

-6.99 (-10.21, -3.77)

-2.80 (-3.67, -1.93)

-2.26 (-5.05, 0.53)

-4.30 (-8.75, 0.15)

-1.59 (-4.03, 0.85)

-0.50 (-1.33, 0.33)

-10.35 (-12.79, -7.91)

-1.36 (-4.37, 1.65)

-6.68 (-8.28, -5.08)

-11.19 (-13.91, -8.47)

-7.36 (-9.41, -5.31)

2.15 (-0.68, 4.98)

-4.33 (-5.83, -2.84)

-1.04 (-3.54, 1.46)

-0.94 (-3.12, 1.24)

-3.30 (-10.80, 4.20)

-3.00 (-4.60, -1.40)

-11.70 (-16.51, -6.89)

-5.30 (-7.80, -2.80)

-9.06 (-12.99, -5.13)

-3.70 (-5.34, -2.06)

difference (95% CI)

-2.50 (-6.01, 1.01)

-1.40 (-2.60, -0.20)

-8.00 (-16.84, 0.84)

1.57 (-1.08, 4.22)

-0.71 (-8.03, 6.61)

-0.68 (-2.14, 0.78)

-3.85 (-9.24, 1.54)

-5.30 (-13.00, 2.40)

-4.59 (-5.11, -4.07)

mean

3.5

3.3

4.2

3.5

2.8

3.7

4.2

3.7

3.4

4.0

3.6

3.8

3.5

3.7

3.8

1.7

4.0

2.6

3.7

3.0

4.0

(%)

3.2

4.1

1.4

3.6

1.8

4.0

2.4

1.7

4.2

weight

AD lower AD higher

0-20 -15 -10 -5 0 5 10

folate

Vitamin B12 Folate

5.64 (3.26-8.02); p < 0.001

4.33 (2.84-5.83); p <0.0.001

35.83 (14.36-57.30); p = 0.001

47.88 (25.01-70.75); p < 0.001

Unadjusted overall

mean difference (95% CI)

Age-adjusted overall

mean difference (95% CI)

Summary and conclusions

• Multiple pathologies associated with AD: synapse loss early

hallmark and a valid target for intervention

• Synapses are composed of neuronal membranes, phospholipids:

possibility for nutritional intervention

• Patients with AD have lower blood and brain levels of key nutrients

with an increased need due to accelerated synapse loss

• Studies of single nutrient supplementation show no benefit

• Dietary patterns suggests combination of nutrients may be

beneficial

• Several studies have demonstrated that Souvenaid increases levels

of key nutrients in plasma

• Recent evidence from MRS suggests enhanced phospholipid

formation

NL-ENIGMA study: Effects of Souvenaid on brain glucose metabolism in early Alzheimer’s disease

• Goal: Exploring the effect of Souvenaid vs. control after 24 weeks on brain glucose metabolism as assessed by 18F-FDG-PET imaging (absolute and relative to a reference region)

• FDG-PET: 18F-FDG-PET assesses glucose metabolism in the brain, which is an index of synaptic function. Both are known to be correlated and impacted in AD

• Population: Subjects with MCI or mild dementia due to AD, stratified for MMSE

• Design:

• Timelines: Last patient out expected in 2017 Supported by grant from NWO (Dutch government)

Decreased brain phospholipids in AD

indicates disrupted membrane integrity

4. Conclusions ……Phospholipids provide an optimal membrane environment for protein interactions,

trafficking and function. There is increasing evidence that phospholipid changes occur

during pathogenic processes in Alzheimer’s disease. …..

Souvenaid increases levels of the

biomarker phospholipids

Baseline and 24-week plasma

samples from the Souvenir II study

Drug-naïve patients with very mild

AD

Polar lipid profile

By providing nutrients which

normally rate-limit phospholipid

synthesis Souvenaid can:

• modify a biomarker profile

reflecting disturbed phospholipid

metabolism

• be useful in asymptomatic

subjects with plasma lipid

biomarker profiles predictive for

conversion to AD

5 / 7 measured phospholipids reported by

Mapstone significantly increased by Souvenaid

* P<0.001; Souvenaid

vs. Control using

ANCOVA

Hartmann et al. JAD, 2014

Mapstone et al. identified a biomarker panel of

10 plasma lipids that can predict conversion

from cognitive healthy to MCI/AD within 2–3

years with >90% accuracy

Changes may reflect the breakdown of neuronal

membranes

Highly publicized findings

set of 10 plasma lipids, including 8 phospholipids

levels are lower in converters and MCI/AD subjects

0,915

0,920

0,925

0,930

0,935

0 12 24

Norm

alise

d p

ath

length

in

beta

band

Time (weeks)

8,20

8,40

8,60

8,80

9,00

9,20

0 12 24

Peak f

requency (

Hz)

Time (weeks)

Control

Active

1,010

1,015

1,020

1,025

1,030

1,035

0 12 24

Norm

alise

d c

lust

eri

ng

coeff

icie

nt

in b

eta

band

Time (weeks)

Control

Active

3. Brain Network Organization* 1. Peak Frequency

p=0.053

p=0.009

2. Phase Lag Index

p=0.011

p=0.019

Network parameters suggest preserved

synaptic formation, function and network

Scheltens et al. J Alzheimers Dis. 2012;31:225-236.

de Waal et al. Plos One 2014;9:1. ITT, MMRM, 2 df contrast, data are mean ±SE

0,12

0,13

0,14

0,15

0 12 24

Phase

Lag Index (

PLI)

in

delt

a b

and

Time (weeks)

ControlActive

1. Basic Quantitative EEG analysis -

Relative power and Peak Frequency

• In AD disturbed signal strength1

1Stam CJ et al. Brain. 2009;132:213-224. 2Stam CJ, van Straaten ECW. Clin Neurophysiol. 2012 doi:10.1016/j.clinph.2012.01.011.

2. Phase Lag Index (PLI) as Functional

Connectivity measure

• In AD loss of PLI1

3. Clustering & Path Length are

measures of Network Organization

• In AD disrupted organization2

PLI

Healthy AD

Electrical activity at the synapse –

EEG: Biomarker for functional connectivity

• Adherence to a Mediterranean diet1,2- higher intake of fish, vegetables,

fruit and monounsaturated fats, can delay cognitive decline

1Scarmeas N, et al . Ann Neurol 2006;59:912–21.2Martinez-Lapiscina EH, et al. J Neurol Neurosurg Psychiatry 2013;84:1318–25.

Epidemiological evidence suggests dietary

patterns can reduce AD risk

Evidence from these and other studies

of dietary patterns suggest that nutrient

combinations are more effective in

reducing AD risk and improving cognitive

outcome than single nutrient intervention

Lower nutrient status in AD preceding classic protein energy malnutrition

Mi et al, Nutrition, 2013, Adapted from Jack et al. Lancet Neurology, 2010

2011- SOUVENIR II: SOUVENAID

IMPROVES MEMORY IN PATIENTS WITH

MILD AD

Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36.

0

0,05

0,1

0,15

0,2

0,25

0 12 24

Mea

n c

han

ge f

rom

bas

elin

e in

N

TB M

emo

ry d

om

ain

z-s

core

Time (weeks)

Control

Active

Significant* (p=0.023) improved

memory

2011- SOUVENIR II: SOUVENAID

IMPROVES MEMORY IN PATIENTS WITH

MILD AD

Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36.

0

0,05

0,1

0,15

0,2

0,25

0 12 24

Mea

n c

han

ge f

rom

bas

elin

e in

N

TB M

emo

ry d

om

ain

z-s

core

Time (weeks)

Control

Active

Significant* (p=0.023) improved

memory

Significant effect on NTB memory

domain during 24 weeks

(whole period trajectory; p=0.023)

Trend on NTB composite score

during 24 weeks

(whole period trajectory; p=0.053)

Statistical analysis re-run by Rush Alzheimer’s Disease Center

ITT, Mixed Model for Repeated Measures (trajectory, mean ± SE)

2011 – Confirmation of improved memory

performance in Mild AD (Souvenir II primary)

Scheltens et al, J Alzheimers Dis. 2012

Change between 24 and

48 weeks (paired t-test):

• p=0.025 active-active

• p=0.008 control-active

2012- Souvenir II open label study