Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR - - PowerPoint PPT Presentation

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Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR - - PowerPoint PPT Presentation

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Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR Spectroscopy G. A. Nagana Gowda 1,2 *, Lauren Abell 2 , Chi Fung Lee 2 , Rong Tian 2 and Daniel Raftery 1,2 * 1 Northwest Metabolomics Research Center and 2 Mitochondria and

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SLIDE 1

Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR Spectroscopy

  • G. A. Nagana Gowda1,2*, Lauren Abell2, Chi Fung Lee2, Rong Tian2 and Daniel Raftery1,2*

1Northwest Metabolomics Research Center and 2Mitochondria and Metabolism Center

Anesthesiology and Pain Medicine, University of Washington, USA

Emails: ngowda@uw.edu and draftery@uw.edu

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SLIDE 2

Graphical Abstract

Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR Spectroscopy

2 GSSG GSH GSH GSSG 8.45 8.50 8.55 8.60 ppm

NAD+ NADP+ NADPH NADH ATP AMP ADP

2 4 6 8 10 12 14 16 18 20 NADH NADPH NADP+ NAD+ AMP 50 100 150 200 250 300 350 ADP ATP GSSG 100 200 300 400 500 600 700 Betaine Serine Acetate 3-… Mannose Creatine Leucine Lysine Glycerol Threonine Valine Glycine Glutami… Proline Alanine Glutamine

Coenzymes Antioxidants

Metabolites

Whole Blood Cardiac Tissue Liver Tissue Brain Tissue Skeletal muscle tissue Kidney Tissue Coenzymes Antioxidants

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SLIDE 3

Abstract: Coenzymes of redox reactions and cellular energy mediate biochemical

reactions fundamental to the functioning of all living cells. To date, no simple method exists to profile these coenzymes in biological samples such as blood and different types of tissue in a single step. Conventional methods that measure them individually carry high risk of errors outweighing their biological variations. We show here that coenzymes including antioxidants and a vast pool of other metabolites can be profiled quantitatively using a simple 1H NMR experiment, simultaneously, in tissue and whole blood. Coenzymes of redox reactions including

  • xidized/reduced nicotinamide adenine dinucleotide (NAD+ and NADH) and

nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH); the coenzymes

  • f

energy including adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and the antioxidants including the sum of oxidized and reduced glutathione (GSSG and GSH) can be measured with essentially no additional effort along with the nearly 70 metabolites that were shown to be quantitated in serum/plasma, recently. Considering that coenzymes and antioxidants represent a sensitive measure of cellular functions in health and numerous diseases, the simple NMR spectroscopy method described here for profiling tissue and blood potentially opens a new avenue for applications in the metabolomics field. Keywords: Redox and energy coenzymes; antioxidants; 1H NMR; quantitation

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SLIDE 4

Introduction

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  • NAD+, NADH, NADP+ and NADPH mediate biochemical reactions fundamental to the

functioning of living cells

  • ATP along with ADP and AMP, represent the cellular energy currency.
  • No simple method exists currently to measure all quantitatively and simultaneously

Conventional analysis method: Enzymatic Assay: Challenges:

  • Separate protocol required for each coenzyme.
  • Interference from sample matrix
  • Finite linear range
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SLIDE 5

Introduction

5

Yes, but… Challenges

  • Ion suppression
  • Interference due to unit mass

difference

  • Interference due to similar

chromatographic retention time

  • In-source fragmentation

Can you Analyze by Mass Spectrometry?

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 + P OH OH O

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 P OH OH O

NADP+ NADPH

N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2 + N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2

NAD+ NADH

N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P O P HO OH O OH O O OH H N N NH2 N N O OH OH O P HO O OH H

ADP AMP ATP

MW=664.4 MW=665.4 MW=744.4 MW= 745.4

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SLIDE 6

Introduction

6

Challenges:

  • Complexity
  • Similar structures
  • Low concentration
  • Instability
  • Enzyme activity
  • Oxidation

Can you Analyze by NMR Spectroscopy?

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 + P OH OH O

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 P OH OH O

NADP+ NADPH

N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2 + N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2

NAD+ NADH

N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P O P HO OH O OH O O OH H N N NH2 N N O OH OH O P HO O OH H

ADP AMP ATP

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SLIDE 7

Results and Discussion Coenzymes in Tissue Extracts

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Heart Kidney Brain Liver Skeletal Muscle

N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2 + N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 + P OH OH O

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 P OH OH O N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2

N N NH2 N N O OH OH O P HO O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P O P HO OH O OH O O OH H

NADP+ NAD+ NADPH NADH ATP ADP AMP

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SLIDE 8

Results and Discussion

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Founding work for coenzymes analysis

Anal Chem. 2015 Jan 6;87(1):706-15

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SLIDE 9

Results and Discussion

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Anal Chem. 2015 Jan 6;87(1):706-15

  • Nearly 70 metabolites

identified and quantitated in human blood

  • ~35% were newly

identified

  • Unprecedented for 1D

NMR.

Founding work for coenzymes analysis

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SLIDE 10

Results and Discussion

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7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 ppm

NAD+ NADP+ NAD+ NAD+ NAD+ NAD+ NADH NADPH ATP/ ADP/ AMP AMP ATP ADP NADH NADPH NADH/NADPH NADP+ NADP+ NADP+ NADP+

9 8 7 6 5 4 3 2 1 ppm 8.45 8.50 8.55 8.60 ppm

NAD+ NADP+ NADPH NADH ATP AMP ADP

Redox and Energy Coenzymes

Mouse Liver Tissue Extract

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SLIDE 11

Results and Discussion: Coenzymes in Different Tissues

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N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2 + N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 + P OH OH O

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 P OH OH O N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2

N N NH2 N N O OH OH O P HO O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P O P HO OH O OH O O OH H

NADP+ NAD+ NADPH NADH ATP ADP AMP

8.45 8.50 8.55 8.60 ppm

NAD+ NADP+ NADPH NADH ATP AMP ADP

Liver Skeletal Muscle Brain Kidney Heart

ATP ATP

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SLIDE 12

Results and Discussion

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8.45 8.50 8.55 8.60 ppm

ATP AMP ADP ATP NAD+ NADH NADPH NADP+

(a)

(b) (c) (d)

The same mouse heart tissue extracted under different conditions

MeOH-Water MeOH-CHCl3, in vivo MeOH-CHCl3

Sensitivity of coenzymes to tissue harvesting/extraction

MeOH-CHCl3, perfusion

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SLIDE 13

Results and Discussion

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8.45 8.50 8.55 8.60 ppm

AMP ATP NADPH NADH NAD+ NADP+

ng/mg tissue

Mouse hearts

100 200 300 400 500

n=6

Simultaneous Analysis of Coenzymes

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SLIDE 14

Results and Discussion

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ng/mg tissue

Mouse hearts

100 200 300 400 500

n=6

Coenzymes from ~5mg tissue

8.42 8.44 8.46 8.48 ppm

80 mg Tissue 20 mg Tissue 5 mg Tissue NAD+ NADPH NADH NADP+ NAD+ NAD+ NADH NADH

* * *

NADPH

*

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SLIDE 15

Results and Discussion

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Coenzymes and Antioxidants in Whole Blood

  • Blood Metabolomics: Serum or Plasma
  • Whole blood: serum/plasma + Cells
  • Nearly 50 % of blood volume is cells and 99% of cells are RBC.
  • Whole Blood Metabolomics:
  • Provides access to wider pool of metabolites with no additional

efforts.

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SLIDE 16

Results and Discussion

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Coenzymes and Antioxidants in Blood

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SLIDE 17

Results and Discussion

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Coenzymes in Whole Blood extract

8.45 8.50 8.55 8.60 ppm

NAD+ NADP+ NADPH NADH AMP ADP

Formic acid

ATP

Whole Blood Blood Plasma

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SLIDE 18

Results and Discussion

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Antioxidants from Whole Blood

2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 ppm

Whole Blood Blood Plasma

GSSG GSH GSSG GSH

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SLIDE 19

Results and Discussion

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8.45 8.50 8.55 8.60 ppm

NAD+ NADP+ NADPH NADH ATP AMP ADP

NAD+/NADH NADP+/NADPH Peak Ratios are Same as Coenzyme Ratios

N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2 + N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 + P OH OH O

N N NH2 N N O O OH O P O OH H N O OH OH O P O OH O O NH2 P OH OH O N N NH2 N N O OH OH O P O OH H N O OH OH O P O OH O O NH2

N N NH2 N N O OH OH O P HO O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P HO OH O O OH H N N NH2 N N O OH OH O P O P O P HO OH O OH O O OH H

NADP+ NAD+ NADPH NADH ATP ADP AMP

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SLIDE 20

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WT mouse heart NAD+ NADH NAD+ NADH WT mouse heart Ndufs4-KO mouse heart NAD+ NADH NAD+ NADH Ndufs4-KO mouse heart

  • Ndufs4 protein critical for mitochondrial function and

ATP synthesis.

  • Ndufs4-KO: NADH/NAD+ ratio

Mice with Ndufs4 gene Knockout

Results and Discussion Biological validation

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SLIDE 21

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  • Increased NAD+ synthesis
  • NAD+/NADH pool

WT mouse heart NAD+ NADH NAD+ NADH WT mouse heart NAMPT-TG mouse heart NAD+ NADH NAD+ NADH NAMPT-TG mouse heart

Overexpression of NAMPT Protein (Nicotinamide phosphoribosyltransferase)

Results and Discussion

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SLIDE 22

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WT mouse heart NAD+ NADH NAD+ NADH

WT mouse heart

NAMPT-TG/Ndufs4- KO mouse heart NAD+ NADH NAD+ NADH

Ndusf4-KO/NAMPT-TG mouse heart

  • Ndufs4-KO: NADH
  • NAMPT-TG : NADH/NAD+ pool

Both NAMPT-TG/Ndufs4-KO

Results and Discussion

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SLIDE 23

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NAD+ NADP+ NADPH NADH Immediately After 24 hours

Degassing

8.40 8.45 8.50 ppm

NAD+ NADP+ NADPH NADH

No degassing

Immediately After 24 hours

Degassing Halts NADH/NADPH Oxidation

Results and Discussion

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SLIDE 24

Results and Discussion

24 Choline Glutamate/ Glutamine Phosphocreatine Phosphocreatine Creatine Creatine Taurine Taurine Glycine Alanine Valine Isoleucine Leucine Lactate Glutamine Glutamate Succinate Acetate Citrate Propane diol Glutamate Glutamate/Glutamine Phenylalanine Tyrosine Fumarate Glucose ATP/ADP NAD+ Histidine NADH

Much More Than the Coenzymes

TCA Cycle Glycolysis Amino acids Carboxylic acids Antioxidants Creatine/Phosphocreatine

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SLIDE 25

Results and Discussion

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2 4 6 8 10 12 14 16 18 20 50 100 150 200 250 300 350 1 2 3 4 5 6 7 8

Blood Plasma Whole Blood

100 200 300 400 500 600 700 10 20 30 40 50 60 70 80 90 100

Coenzymes/Antioxidants

1000 2000 3000 4000 5000

Concentration (µM) Concentration (µM)

Concentrations of coenzymes, antioxidants and

  • ther metabolites in human blood
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SLIDE 26

Conclusions

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  • We provide a simple NMR method for simultaneous quantification of coenzymes
  • f cellular redox reactions and energy in tissue and blood.
  • We demonstrate importance of harvesting/extraction methods for reliable

analysis, as many coenzymes can evade detection wholly or partly depending on the method.

  • We show that over a period of time NADH and NADPH levels diminish due to
  • xidation in solution; however, degassing can halt such a change.
  • Most of the coenzymes can be measured using as little as 5 mg tissue and a

further improvement in the detection limit can be achieved using sensitivity enhancement methods such as using micro-coil probes.

  • The ability to measure coenzymes and antioxidants along with a vast pool of
  • ther metabolites using NMR potentially opens a new chapter in the

metabolomics field.

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SLIDE 27

Supplementary Materials

Links: http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b00171 http://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b00442 http://pubs.acs.org/doi/abs/10.1021/ac503651e

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SLIDE 28

Acknowledgments

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Funding:

Royalty Research Fund (RRF)