A. The LIPID MAPS Initiative B. Example: Macrophage Eicosanoids

D. Human Plasma Lipidome and Biomarkers

Edward A. Dennis Departments of Chemistry/Biochemistry and Pharmacology

School of Medicine, University of California, San Diego

LIPID MAPS “Glue Grant” NIH U54 GM 69338

PERCEPTION OF THE LIPIDOME AFTER THE FIRST DECADE OF LIPIDOMICS

Lipidomics Joins the Omics Evolution (2009), Dennis, PNAS, 106, 2089.

4 base side chains

20 amino acid side chains

nucleic acids, amino acids, sugars and fats: >105 ?

all the fats: >105 ?

Increase in Omics Citations 1985-2009

Wenk MR (2010) Lipidomics: New Tools and Applications. Cell, 143, 888-895. Year

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Saccharolipids / Polyketides

probe

medium cells tissues

homogenate

sonicate

internal standards

extraction

cells or tissues

extract

GC LC

3. Additives (for ionization) 4. Mass spectrometer monitoring modes

(variables) 1. Mass spectrometer types 2. Ionization mode

mass spectrometer

“CLASS”: Comprehensive Lipidomics Analysis using Separation Simplification

category specific (deuterated, odd-chain carbon)

category optimized (liq-liq, SPE)

category specific (GC, NP-HPLC, RP-HPLC, chiral, specialty)

a “divide-and-conquer” strategy

Harkewicz & Dennis, “Applications of mass spectrometry to lipids and membranes” Ann Rev Biochem, 80: 301-325 (2011)

Nuclei – DAPI Mitochondria – MitoTracker Red

Kdo2-Lipid A (KLA, LPS subspecies) A specific agonist of TLR-4 on RAW 264.7 macrophages

Raetz et al., 2006, J. Lipid Res. 47: 1097

O-Specific chain

Core

Lipid A

Glycophospholipid

Polysaccharide

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Dennis et al (2010) J. Biol. Chem,  51, 39976-85

Buczynski, Dumlao, Dennis (2009) JLR, 50, 1015-38

Cellular Eicosanoid Metabolism

5-LOX

12-LOX

CYP(EET) 15-LOX

COX CYP(ω)

Cellular eicosanoid metabolism

Buczynski, Dumlao, Dennis, 2009, JLR, 50: 1015-38

Cellular eicosanoid metabolism

COX

Directed Proteomics on Enzymes of Eicosanoid Biosynthesis

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Sabido, Quehenberger Dennis, Aebersold (2012) Mol Cell Proteomics 11: M111.014746

Quehenberger & Dennis, New Eng. J. Medicine, 365, 1812-23 (2011)

Eicosanoid Genes to Proteins to Metabolites Proteins

The Beneficial Lipids in Fish Oil •  Fish  oil  is  composed  primarily  of  two  ω-­‐3  PUFAs:  

•  Humans  synthesize  from  essen=al  fa>y  acid  α-­‐linolenic  acid  (C18:3,  ω-­‐3)  

•  EPA  is  found  at  barely  detectable  levels  in  humans  

•  DHA  is  more  abundant  than  EPA;  found  primarily  in  the  brain  and  re=na    

Eicosapentaenoic  Acid  (EPA)  (C20:5,  ω-­‐3)  

Docosahexaenoic  Acid  (DHA)  (C22:6,  ω-­‐3)  

ω-­‐3  

ω-­‐3  

ω  

ω  

Arachidonic Acid Serves as a Precursor for Many Eicosanoids

Are eicosanoids less pro-inflammatory when derived from EPA/DHA?

EPA/DHA ?

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Effects of PUFA Supplementation on ATP Stimulated COX-1 and 5-LOX Signaling

Norris & Dennis, PNAS (2012) 109, 8517

Omega-3 Implications for Nutrition •  The global effects of EPA and DHA on normal lipid

metabolism can be quantitatively studied.

•  EPA and DHA affect the overall eicosadome decreasing production of some, but not all, AA-derived eicosanoids.

•  There is a concomitant increase in specific EPA- and DHA-derived metabolites.

•  Deciphering the role of fish oil-derived ω-3 EPA and DHA in inflammatory eicosanoid signaling provides insight as to their role as therapeutic/nutritional agents.

Norris & Dennis, PNAS (2012) 109, 8517

NIST collected (pooled) fasting plasma from 100 individuals 50% female and 50% male; age 40-50

15% of the total taken from individuals of Hispanic origin!

Nucleic Acids

Amino Acids

Sugars

Lipids

Human Plasma Metabolites (mg/dL)

Glycerolipids

Fatty Acyls

Glycerophospholipids

Sphingolipids

Sterol Lipids

Prenols

Human Plasma Lipid Categories (µM)

Human Plasma Lipid Diversity

J Lipid Res, 51, 3299-3305 (2010)

Plasma Lipids in the Metabolic Syndrome

Quehenberger & Dennis, New Eng. J. Medicine, 365, 1812-23 (2011)

Alex Andreyev Howard Hsu Oswald Quehenberger Paul Norris

Darren Dumlao Ann Gregus Jian Cao Aaron Armando Ishita Shah

Varnavas Mouchlis Matt Buczynski John Burke Yasu Kihara

Eoin Fahy S. Subramaniam

Charles Brown Univ. of Missouri

George Kokotos Univ. of Athens

Tony Yaksh

Chris Glass

Kang Zhang

Eduard Sabidó

Ruedi Aebersold

Andy McCammon

Virgil Woods

The LIPID MAPS Consortium L-R: C.R.H. Raetz, J.L. Witztum, N. Winograd, A.H. Merrill, R.C. Murphy, M.S. VanNieuwenhze, E.A. Dennis, D.W. Russell, W.A. Shaw, C.K. Glass, J. Chin (NIH), S. Subramaniam, H.A. Brown