Integrated Approaches to Translational Redox Biology and Bioenergetics

Cellular

Metabolism

Molecular

Metabolism

Trusted answers

that advance the quality of life

For Research Use Only. Not for diagnostic purposes.For Research Use Only. Not for diagnostic purposes.

Changing the way metabolism is measured

Mitochondria

Fatty Acid Oxidation

Glycolysis

For Research Use Only. Not for diagnostic purposes.For Research Use Only. Not for diagnostic purposes.

Agilent Seahorse is the leader in cellular energy metabolism

>1,300 instruments >10,000 users

>2,400 publications (Jan 2016)

232

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600

783

1 7 16 41 103

240

472

881

1,481

2,264

0

500

1000

1500

2000

2500

2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

Publications Cumulative

For Research Use Only. Not for diagnostic purposes.For Research Use Only. Not for diagnostic purposes.

A routine measure of bioenergetic health is needed

Clinical trials

Phase I-IV* 2000-2004 2005-2009 2010-2014

Mitochondrial targets 7 86 114

Metabolic targets 18 225 350

Neurodegeneration: Parkinson’s, Huntington’s, Alzheimers

diabetes mellitus

hepatic failure

Cancer

autoimmunityGVHD Renal disease:

proximal tubulopathy

Cardiovascular disease:*From NIH ClinicalTrials.gov database

What is needed is a simple 1 hour blood test

Victor Darley-Usmar, PhDProfessor UAB

Translational MedicineClinical diagnostics

David Ferrick, PhDSr. Dir. Agilent

Laura Stanley, FMM

Aging

Genetic Factors Lifestyle and

Environmental

Diabetes

Obesity

Cancer

Neurodegeneration

AtherosclerosisInfection

Inflammation

Energetics, Pathology and Redox

What is healthy and how does it Change in disease?

Isolated Mitochondria and Cellular Mitochondria

Excess substrate (usually for one Complex only).

Saturating ADP.

State 3 until ADP is depleted, then State 4.

Substrate not saturating.

Low ADP.

Regulated and intermediate

Networked and Dynamic

The Mitochondrial Electron Transport Chain

Q

Complex I(NADH

Dehydrogenase)

Complex II(Succinate

dehydrogenase)

Complex III(Ubiquinol-

cytochrome coxidoreductase)

Complex IV(Cytochrome

c oxidase)

Complex V(ATP

synthase)

e-

e-

O2 H2O

H+ H+ H+

H+

H+

H+

H+

H+

ADP + Pi ATP

Rotenone TTFA MyxothiazolAntimycin A

CyanideNO˙

Oligomycin

Inhibitors

How can we measure mitochondrial function in intact cells?

OxygenConsumption Rate

(OCR)

=Mitochondrial

Respiration

ExtracellularAcidification Rate

(ECAR)

=Glycolysis

Measuring Mitochondrial Function

CII

-AD

P

St

3

St 4

CII

-FC

CP

Measuring Mitochondrial Function in Cells

PMP

Succ

Rot

ADP

Oligo Ant-A

0

20

40

60

80

100

120

140

160

180

200

0 20 40 60 80 100

OC

R (

pm

ol/m

in)

Time (min)

HV

FCCP

H+

ADP ATP

H+

H2OO2

X

H2OO2

H+

H+

FCCP

X

RCR =State 3

State 4

Measuring Mitochondrial Function in Cells

0

1

2

3

4

5

6

7

Platelets Mono

Sta

te3

/sta

te 4

RCR (CII)

0

50

100

150

200

250

300

350

CI-F CIV-F CII-F CII-ADPO

CR

(p

mo

l/m

in) Platelets

0

20

40

60

80

100

120

140

160

CI-F CIV-F CII-F CII-ADP

OC

R (

pm

ol/

min

)

Mono

FCCP

BASAL

ATP

LEAK

MAXIMAL

NON-MITOCHONDRIAL

Letting the cell drive its own mitochondria

200

300

400

500

100

0

Time

OligomycinRESERVE CAPACITY

H+

ADP ATP

H+

H2OO2

X

H2OO2

H+

H+

FCCP

FCCP

Antimycin A

OC

R (

pm

ol O

2/m

in)

X

MAXIMAL

Cell fuel

Ma

xim

al

Non-Mito

Ma

xim

al

Non-Mito

Basal

Basal

Comparing Mitochondrial Activity to CellularBioenergetics

O

FAA

0

20

40

60

80

100

120

140

160

CI-F CII-F CIV-F

OC

R (

pm

ol/

min

)

Mono

0

50

100

150

200

250

300

350

CI-F CII-F CIV-F

OC

R (

pm

ol/

min

)

Platelets

O

F AA

0

50

100

150

200

250

300

350

0 20 40 60 80

OC

R (

pm

ol/

min

)

Time (min)

Platelets

0

20

40

60

80

100

120

140

160

0 20 40 60 80

OC

R (

pm

ol/

min

)

Time (min)

Mono

Mitochondria Cellular Bioenergetics

The energy profile represents a fundamental bioenergetic program; a clinical test?

0

50

100

150

200

250

300

350

0 10 20 30 40

Oxy

gen

Co

nsu

mp

tio

n R

ate

(pm

ol O

2/m

in/μ

g p

rote

in)

Time (min)

Cardiomyocytes

F

A+R

0

20

40

60

80

100

0 20 40 60

Oxy

gen

Co

nsu

mp

tio

n R

ate

(pm

ol O

2/m

in/μ

g p

rote

in)

Time (min)

Hepatocytes

F

O

A+R

0

10

20

30

40

0 20 40 60

Oxy

gen

Co

nsu

mp

tio

n R

ate

(pm

ol O

2/m

in/μ

g p

rote

in)

Time (min)

Endothelial Cells

FO

A

O

BasalEnergy

Demand

The Bioenergetic Health Concept

Bio

en

erg

etic

He

alth

Unhealthy

IncreasedATP Demand

Cumulative effects of Disease and therapy on bioenergetic health

Unhealthy

Bio

en

erg

etic

Fu

nct

ion

BasalenergeticDemand

BioenergeticDysfunction

Basal

Non-Mitochondrial

Maximal

Reserve Capacity

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 5 10 15 20 25 30 35 40 45

OC

R(p

mo

les

/min

/µg

pro

tein

)

Time (min)

ATP

Linked

H+ Leak

We can use the profile to measure the bioenergetic health index (BHI)

O F A

BHI = (Reserve Capacity) x (ATP linked)

(H+ Leak) x (Non-mitochondrial)

BHI = (Reserve Capacity) x (ATP linked)

(H+ Leak) x (Non-mitochondrial)

BHI = (Reserve Capacity) x (ATP linked)

(H+ Leak) x (Non-mitochondrial)

BHI = (Reserve Capacity) x (ATP linked)

(H+ Leak) x (Non-mitochondrial)

BHI = (Reserve Capacity) x (ATP linked)

(H+ Leak) x (Non-mitochondrial)

0

2

4

6

8

10

12

0 20 40 60 80 100

OC

R (

pm

ol/

min

/µg

pro

tein

)

Time (min)

Control MNC

15 µM HNE

20 µM HNE

25 µM HNE

30 µM HNE

The Profile gets worse with stress

Unhealthy

Bio

en

erg

etic

Fu

nct

ion

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

0 10 20 30

BH

I

OC

R (

pm

ole

/min

/µg

pro

tein

)

HNE (µM)

Reserve CapacityBHI

BHI is a sensitive index of oxidative stress

The mitochondrial stress profile is an integrated response.

What do we use to measure this?

Unhealthy

Bio

en

erg

etic

Fu

nct

ion

Muscle Biopsy

Venupuncture

Translational Bioenergetics

Blood leukocytes-and plateletssensors of bioenergetic health…..?

Exposed to the soluble factors of circulation

Interaction with multiple organs/tissues in the body

Respond to changes in the body (physiological/pathological)

Mediate the process of inflammation

Energy mapping of leukocyte and platelets

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6

OC

R (

pm

ole

s/m

in/µ

g p

rote

in)

ECAR (mpH/min/µg protein)

Lymph

Plt

Mono

Neutro

Highly Energetic

Glycolytic

Oxidative

Low Energy

• PBMCs are a mixed population with different metabolic programs

Mitochondrial defect in patients

0

10

20

30

40

50

60

70

80

Healthy Mitochondrial Disease

Bio

en

erge

tic

Hea

lth

In

dex

#

Bioenergetic Health Index

0

1

2

3

4

5

6

7

Complex -I Complex-IV

Healthy

Mito Disease

OC

R(p

Mo

l/m

in/1

0,0

00

cel

ls)

#

Mitochondrial Respiratory Complexes

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Basal ATP-Linked Maximal Reserve Capacity

Healthy

Mitochondrial Disease

OC

R(p

Mo

l/m

in/1

0,0

00

cel

ls)

#

#

Bioenergetic Parameters

Monocytes

38 year old MaleEpisodes of extreme fatigue

Mitochondrial Function in PBMC’s as a marker for other tissues

Vervet Primate Model of AgingSusceptible to frailty and sarcopenia

*Cohort represents a wide range of ages and metabolic health status

Anthony Molina: Wake Forest School of MedicineRedox Biology, 2016

Anthony Molina: Wake Forest School of MedicineRedox Biology, 2016

Skeletal MuscleFibers

Skeletal MuscleMitochondria

Monocyte energetics vs skeletal muscle (vastus lateralis)

HIV Metabolism: Then and Now

Nucleoside RT inhibitors“D-drugs”- ddI, d4T, ddC Lipodystrophy Lactic Acidosis Neuropathy Hepatic steatosis Known Mitochondrial toxicity Sometimes fatal

A disease of metabolic stress

Unknown long term toxicity 50+ years of therapy?

Few studies addressing mitochondrial toxicities Protease inhibitors, non-nucleoside

RT inhibitors

Boosting agents-ritonavir, cobicistat

Total Body Fat Decreases BHI in HIV Patients

Adjusted for age, racesmoking

r = -0.37

p = 0.03

BH

I

female; ages 19-55;white or African-American body mass index (BMI) ≥ 18.5; HIV viral load < 400 copies/mL; dual-energy x-ray absorptiometry (DXA); prescribed ART regimen for less than 10 years

AVR, MVR CABG

Aortic/Mitral Valve replacement or repair Coronary artery bypass graft surgery

• Increasing incidence of coronary and valvular heart disease.

• More than 500,000 CABGs and 100,000 AVR/MVRs performed each year.

• Postoperative complications include arrhythmias, stroke, Myocardial infarction, respiratory distress and acute renal failure.

Cardiothoracic surgery

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 10 20 30 40

Co

nc

en

tra

tio

n (

µg

/mL

)

Hour Post-Surgery

CK-MBPeric. Fluid

Blood Serum

The postoperative pericardial environment

Mediastinal drainage tubes

Heart

Blood PCF

Hemolyzedsupernatant

Plasma

WBCs

Packed Red Blood Cells

Lipid layer

0

2

4

6

8

10

12

14

16

18

HealthyControls

Post-opBlood

Post-opPCF

BH

I

*

**

0

2

4

6

8

10

12

Post-op Blood Post-op PCF

BH

I

The BHI in postoperative cardiac surgery patients

The next stage integration with metabolomics

65% common to 2 out of 3 donors

0 10 20 30 40 50

0

600

1200

1800

m/z

Retention time (min)

NP-NEG

Fatty acyls

Glycerolipids

Glycerophospholipids

Polyketides

Prenol lipids

Saccharolipids

Sphingolipids

Sterol lipids

Other metabolites

Unknown

0 5 10 15 20 25 30

0

600

1200

1800

m/z

Retention time (min)

Polar-NEG

The resting platelet lipidome: Up to 8000 Lipids

D3

510

561

515

619

3539

526

1807

D1 D2

0 5 10 15 20 25 30

0

600

1200

1800

m/z

Retention time (min)

Polar-NEG

Num

be

r of

hits

FA GL PL PK PR SA SP SL OM

164

565

1075

72 616

157

49

397

0

300

600

900

1200Total number of lipids

Donor 1 5229

Donor 2 5141

Donor 3 6491

Mean ± SEM 5620 ± 436

≥ 2 of 3 donors 5245

Total 8077

A B C

D

E F

0 5 10 15 20 25 30

0

600

1200

1800

m/z

Retention time (min)

Polar-POS

0 10 20 30 40 50

0

600

1200

1800

m/z

Retention time (min)

NP-NEG

0 10 20 30 40 50

0

600

1200

1800

m/z

Retention time (min)

NP-POS

Fatty acyls

Glycerolipids

Glycerophospholipids

Polyketides

Prenol lipids

Saccharolipids

Sphingolipids

Sterol lipids

Other metabolites

Unknown

Figure1FA: Fatty acyls

GL: Glycerolipids

PL: Glycerophospholipids

PK: Polyketides

PR: Prenol lipids

SA: Saccharolipids

SP: Sphingolipids

SL: Sterol lipids

OM: Other metabolites

FA: Fatty acyls

GL: Glycerolipids

PL: Glycerophospholipids

PK: Polyketides

PR: Prenol lipids

SA: Saccharolipids

SP: Sphingolipids

SL: Sterol lipids

OM: Other metabolites

D3

160

68

150

138

439

108

487

D1 D2

5138 107 646

Control Thr-Upreg

Effect of thrombin on basal lipids in ≥ 2 donors

Total number of lipids

Donor 1 805

Donor 2 765

Donor 3 1172

Mean ± SEM 914 ± 130

≥ 2 of 3 donors 753

Total 1550

G

0 10 20 30 40 50

0

600

1200

1800

m/z

Retention time (min)

NP-POS

Oxidised phospholipids

0 10 20 30 40 50

0

600

1200

1800

m/z

Retention time (min)

NP-NEG

Oxidised phospholipids

H

A subset of lipids are selectively upregulated in

thrombin-activated platelets

Aspirin blocks generation of many platelet lipids but increases 5% in

some people

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

18:0p/22:5(O)-PE

16:0a/HETE-PE

18:0p/22:4(O)-PE

18:0a/22:5(O)-PE

16:0a/22:5(O)-PE

18:1p/22:4(O)-PE 16:0a/22:4(O)-PE

16:0p/DXA3-PE 18:2a/HETE-PC 18:0p/20:4(3O)-PE

18:2a/HETE-PE 18:0a/HDoHE-PE

18:1a/22:4(O)-PE 18:0a/DXA3-PE 18:0p/20:4(2O)-PE 16:0p/20:4(2O)-PE 18:1p/DXA3-PE

18:1p/20:4(3O)-PE

18:0a/20:4(3O)-PE

18:1a/HDoHE-PE

20:1a/12-HETE-PE

16:0p/20:4(2O)-PE

18:0p/HETE-PE

16:0p/22:5(2O)-PE

0 10 30 120 (min)

Scalebar:Log10forintegratedarea(cps)foreach

lipidmeasuredasparenttosn2fa; yacid

carboxylateanion.

Figure3

A

Rela

tive

Ab

un

da

nce

14-HNTrE (n-6) m/z 307.2282

150 200 250 300

207.1389

263.1649

0

50

100 289.2169

-C6H12O

m/z

14-HNDE (n-6) m/z 309.2438

150 200 250 300

209.1547

291.2328

265.1810 0

50

100

-C6H12O

m/z

Rela

tive A

bun

da

nce

14-HNTE (n-6), m/z 305.2126

+ Malondialdehyde, m/z 71.0133

DPA; 22:5, m/z 329.2486

Δ4 dihomo-TXA2 ; 22:5 (+3O), m/z 377.2333

COX-1, TXA2-synthase

C14-HNTE (n-6)

14-HNTE (n-3)

14-HNTrE (n-6)

14-HNDE (n-6)

205.1235

207.1391

207.1389

209.1547

14-HNTE (n-3)

m/z 305.2126

m/z

150 200 250 300

0

50

100 141.0171

287.2014

261.2224

207.1391

-C6H10O

Re

lative

Abu

nd

an

ce

150 200 250 300

141.0171

287.2014

205.1235

0

50

100

261.2224 -C6H12O

m/z

14-HNTE (n-6) m/z 305.2126

Rela

tive

Abu

nd

an

ce

B

NR NR

340

379

34

DThrombin upregulated ≥ 2 out of 3 donors effect of aspirin

FA: Fatty acyls

GL: Glycerolipids

PL: Glycerophospholipids

PK: Polyketides

PR: Prenol lipids

SA: Saccharolipids

SP: Sphingolipids

SL: Sterol lipids

OM: Other metabolitesE

Nu

mb

er

of

hits

FA GL PL PK PR SA SP SL OM

55

17

22

1310

13 2

51

0

20

40

60

700+ Lipids increased in response to thrombin

Eicosanoids levels are dependent on mitochondrial function during platelet activation

Thrombin

Thrombin

Aspirin-COX-2 controls Fatty Acid Metabolism In Platelets

ControlAspThr + Thr + Asp

What is normal-bioenergetics?M

aking

Ene

rgy

Mitochondrial Activity

What is normal-metabolism

Donor 1 Donor 2 Donor 3 Donor 4 Donor 5

-4

-2

-2

-4

0

5 Stored Platelets(Day 6)

300x106 platelets

Acetonitrile: Water (2:1)

LC-Orbitrap MS

Manhattan Plots(xmsPanda analysis)

Hierarchal Clustering

11,173 total m/z’s detected

324 significantly decreased features;

92 significantly increased features

Pathological Stress

Max

imal

Mit

och

on

dri

al F

un

ctio

nCan we improve BHI in Human Populations

with Mitochondrial Therapeutics?

BasalMitochondrial Demand

Reserve Capacity

SusceptibilityTherapy

Metabolotranscriptoprofile

http://www.the-aps.org/mm/Conferences/APS-Conferences/2017-Conferences/Bioenergetics